There is widespread use of ionization sources (ambient and non-ambient) for a variety of applications. More recently, charged microdroplets generated by electrospray ionization and paper spray have been used to conduct chemistry at faster rates compared to bulk volumes. Uncharged droplets such as those generated by the Leidenfrost technique have also been used to explore chemistry and study the degradation of drugs in an accelerated manner. These microdroplets serve as reaction vessels in which in which some reactions are known to occur at accelerated rates. Such chemistry can be particularly useful in pharmaceutical settings to rapidly synthesize small amounts of materials in relatively short amount of time. Additionally, microdroplets may also be used to perform high throughput screening analysis. While several parameters influencing the rate of reaction in microdroplets have been explored (such as spray distance and reagent concentration), the mechanism of reaction acceleration has not been probed to a significant extent. A major portion of my dissertation describes the use of charged and uncharged microdroplets to perform quick chemistry, guide microfluidic synthesis of drugs such as diazepam, perform scale up of copper catalyzed C-O and C-N coupling reactio<a></a>ns and screen reaction conditions for pharmaceutically relevant reactions such as the Suzuki cross-coupling reaction. Additionally, work discussed here also describes development and use of existing techniques such as structured illumination microscopy to measure droplet sizes, explore the role of distances on droplet size, and study the effect of surfactants on the rate of reactions in microdroplets generated by nano-electrospray ionization. A mathematical model to understand the mechanism of increased reaction rates in microdroplets has also been presented. Additionally, this dissertation also describes ways to manipulate ions in air using various designs of 3D-printed electrodes that operate with DC potentials only and which can be easily coupled with nano-electrospray ionization sources to transmit ions over long distances

New experimental strategies and algorithmic approaches were devised and tested to improve the analysis of pharmaceutically relevant materials. These new methods were developed to address key bottlenecks in the design of amorphous solid dispersions for the delivery of low-solubility active pharmaceutical ingredients in the final dosage forms exhibiting high bioavailability. <br>

Although seasonal influenza epidemics represent a significant threat to public health, their treatment options remain limited. With deaths from the 1918 influenza pandemic estimated at >50,000,000 worldwide and future pandemics predicted, the need for a potent broad-spectrum influenza therapy is critical. In this thesis, I describe the use of a structurally modified zanamivir, an influenza neuraminidase inhibitor that blocks the release of nascent virus, to deliver attached therapeutic agents specifically to the surfaces of viruses and virus-infected cells, leading to simultaneous inhibition of virus release and immune-mediated destruction of both free virus and virus-infected cells. Chapter 1 describes the major characteristics of the influenza virus, the morbidity and mortality associated with annual infections by current strains of the virus, and the treatments available to reduce the disease burden associated with these infections. Chapter 2 describes the design, synthesis, and evaluation of a zanamivir-related targeting ligand and its conjugation to two orthogonal imaging agents which are then used to characterize the binding specificity and biodistribution of the targeting ligand in influenza virus-infected cells and in infected mice. Chapter 3 describes the development of an influenza virus-targeted immunotherapy, where a zanamivir-targeted hapten is exploited to redirect the immune system to destroy influenza virus and virus-infected cells. When tested in vivo, this immunotherapy is shown to be significantly superior to zanamivir in protecting mice from lethal influenza virus infections. Finally, both a zanamivir-targeted chemotherapy and a CAR-T cell therapy with different mechanisms of cytotoxicity against neuraminidase expressing cells are introduced in Chapter 4.

A thorough understanding of pharmaceutical and therapeutic products and materials is important for an improved quality of life. By probing the complex behaviors and properties of these systems, new insights can allow for a better understanding of current treatments, improved design and synthesis of new drug products, and the development of new treatments for various health conditions. Often, the impact of these new insights are limited by current technology and instrumentation and by the methods in which existing data is processed. Additionally, current standards for characterization of pharmaceuticals and therapeutics are time-consuming and can delay the timeline in which these products become available to the consumer. By addressing the limitations in current instrumentation and data science methods, faster and improved characterization is possible.<div><br></div><div>Development and improvement in optical instrumentation provides potential solutions to the current limitations of characterization methods by conventional instrumentation. Limitations in speed can be addressed through the use of nonlinear optical (NLO) methods, such as second harmonic generation (SHG) and two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy, or by linear methods such as fluorescence recovery after photobleaching (FRAP). For these methods, a high signal-to-noise ratio (SNR) and a nondestructive nature decrease the overall sample size requirements and collections times of these methods. Furthermore, by combination of these optical techniques with other techniques, such as thermal analysis (e.g. differential scanning calorimetry (DSC)), polarization modulation, or patterned illumination, the collection of more complex and higher quality data is possible while retaining the improved speed of these methods. Thus, this modified instrumentation can allow for improved characterization of properties such as stability, structure, and mobility of pharmaceutical and therapeutic products.<br></div><div><br></div><div>With an increase in data quantity and complexity, improvements to existing methods of analysis, as well as development of new data science methods, is essential. Machine learning (ML) architectures and empirically validated models for the analysis of existing data can provide improved quantification. Using the aforementioned optical instrumentation, auto-calibration of data acquired by SHG microscopy is one such method in which quantification of sample crystallinity is enabled by these ML and empirical models. Additionally, ML approaches utilizing generative adversarial networks (GANs) are able to improve on identification of data tampering in order to retain data security. By use of GANs to tamper with experimentally collected and/or simulated data used in existing spectral classifiers, knowledge of adversarial methods and weakness in spectral classification can be ascertained. Likewise, perturbations in physical illumination can be used to ascertain information on classification of real objects by use of GANs. Use of this knowledge can then be used to prevent further data tampering or by improving identification of data tampering.<br></div>

<p>Mass spectrometry, the science and technology of ions, owes much of its current popularity to the development of electrospray ionization. The development of electrospray ionization, along with its low flow-rate analog nanoelectrospray ionization, has increased the chemical space that can be investigated using mass spectrometers by orders of magnitude. While the interfacial chemistry of charged microdroplets that are generated by nanoelectrospray has been studied in detail, the physics of their motion, particularly in the presence of an applied field at ambient pressures, remains relatively unexplored. In this dissertation, an increase in ion currents detected by a commercial triple quadrupole mass spectrometer is used to demonstrate that: (i) the orthogonal injection of counterions into an electrode assembly can compensate for space charge effects and enhance the sampling of charged microdroplets from a nanoelectrospray focused electrostatically under ambient conditions into the mass spectrometer; and (ii) the ease of ion evaporation from charged microdroplets may be elucidated for small molecules based on their relative transmission through an electrode assembly for the simultaneous ambient electrostatic focusing of two nanoelectrosprays. In each case, the development is characterized by using ion trajectory calculations in conjunction with experiments, using homebuilt devices designed and fabricated in-house as rapid prototypes via 3D printing. In the open air, charged microdroplets have low kinetic energies with a narrow energy spread. Despite these limitations, this dissertation demonstrates, through the electrostatic manipulation of charged microdroplets produced via nanoelectrospray ionization, that a better understanding of the physics of moving charges in the open air can be used to increase the sensitivity of atmospheric pressure ionization.</p>

<p dir="ltr">Originating from J. J. Thomsons original work and the development of electrospray ionization (ESI) by John B. Fenn, mass spectrometry offers a versatile analytical tool to measure beyond an ion’s m/z, especially for biomolecules. Gas-phase ion/ion reactions within a mass spectrometer offers an attractive approach to study biomolecules as they take place on the millisecond and sub millisecond time scale, have high efficiency, allow oppositely charged ions to interact with each other in a controlled manner, and a allows for selection of each reactant prior to the reaction via ion isolation. This can be used to probe gas-phase chemistry that can reflect reactions in solution, however gas-phase reactions have no solvent effects and happen faster, making it a simpler experiment. Here, a variety of gas-phase ion/ion reactions and ion trap methodologies are described to study mostly lipids with a minor amount of transmetalation at the beginning.</p><p dir="ltr">First, a series of multivalent metals complexed to neutral ligands are demonstrated to form ion-pairs with tetraphenylborate anions via ion/ion reactions. The resulting products were subjected to collision induced activation (CID) to observe their involvement in transmetalation, complementary density functional theory (DFT) calculations are provided as well. Next, sequential ion/ion reactions were performed to convert isomeric phosphoinositol phosphates dianions to monocations to reveal structural characterization and isomeric differentiation utilizing tandem MS and dissociation kinetics. The following two chapters after, reports on complementary efforts to separate lipids in the gas-phase of different mass and charge but similar mass-to-charge (m/z) resulting in overlapping m/z signals. The first report demonstrates a physical approach where singly and double charged lipids are separated in space from each other, trapped simultaneously such that no information is lost. The second utilizes a lanthanide, Yb3+ trication complex that underwent ion/ion reactions with singly and doubly charged lipid anions of similar m/z that result in different m/z products for each singly and doubly charged lipids. Lastly, a sequential ion/ion approach utilizing hexa(ethylene glycol) dithiol as a novel reagent to charge invert structurally uninformative lipid cations to structurally informative anions with subsequent carbon-carbon double bond localization.</p>

<p>The thesis consists of two main parts of nonlinear optical instrumentation development. </p> <p>Fluorescence-detected mid-infrared photothermal (F-PTIR) microscopy is demonstrated for sub-diffraction limited mid-infrared microspectroscopy of model systems and applied to probe phase transformations in amorphous solid dispersions. To overcome the diffraction limit in infrared imaging, a highly localized temperature-dependent photothermal effect is an attractive alternative indicator to infrared absorption. Photothermal atomic force microscopy infrared spectroscopy (AFM-IR) achieves nanometer resolution by monitoring heat caused expansion but only restricted on the surface. For 3D imaging, optically detected photothermal infrared (O-PTIR) combines an infrared laser with a visible probe source with to transduce photothermal refractive index changes (e.g., from changes in beam divergence or scattering). The sensitivity of O-PTIR is ultimately limited by the relatively weak dependence of refractive index with temperature, exhibiting changes of ~0.01% per oC. Fluorescence-detected photothermal mid-infrared (F-PTIR) spectroscopy (Fig. 1) is demonstrated herein to support 3D imaging with improved photothermal sensitivity. In F-FTIR, the sensitivity of fluorescence quantum efficiency to temperature change (~1-2% per oC) is used to transduce transient heat flux from localized IR absorption. The infrared spatial resolution of F-FTIR is defined by fluorescence microscopy and the thermal diffusivity of the sample instead of infrared wavelength. Initial F-PTIR proof of concept studies are described for microparticle assemblies of silica gel and polyethylene glycol, followed by applications of F-PTIR for analysis of localized composition within phase-separated domains induced by water vapor exposure of an amorphous solid dispersion (ritonavir in copovidone).</p> <p>Fluorescence recovery while photobleaching (FRWP) is demonstrated as a method for quantitative measurements of rapid diffusion mapping over the microsecond to millisecond time scale. Diffusion measurements are critical for molecular mobility assessment in cell biology, materials science and pharmacology. Fluorescence recovery after photobleaching (FRAP) is a well-known noninvasive optical microscopy method for measuring diffusion coefficients of macromolecules, such as proteins in cells and viscous solutions. However, conventional point-bleach FRAP is challenging to implement with multi-photon excitation and typically only supports diffusion analysis over millisecond time scales due to camera frame rate limitations. FRWP with patterned illumination addresses these limitations of FRAP by probing the fluorescence intensity changes while bleaching a comb pattern within a field of view (FoV). Fast-scanning of an ultrafast excitation beam distributes heat rapidly over multiple adjacent pixels, minimizing local heating effects that could complicate analogous diffusion measurements by point-bleach FRAP with multiphoton excitation. In FRWP, time-scales of the probed diffusion events are defined by a single line-pass time of a resonant scanning-mirror with a period of 125  s. In FRWP, the bleach pattern spans locations across the whole FoV, enabling diffusion mapping through image segmentation. More than a hundred bleaching and recovery events can be recorded during a single 10s measurement. Normal and anomalous diffusion of rhodamine-labeled bovine serum albumin (BSA) molecules was studied as a model system, with applications targeting rapid assessment of therapeutic macromolecule mobility within heterogeneous biological environments.</p>

<p dir="ltr">Metal ions are typically considered a nuisance for mass spectrometry, as they can introduce chemical noise and distribute an analyte’s signal into multiple peaks. In some cases however, metal ions in biological solutions are either necessary for biomolecular structures, or so ubiquitous in a sample’s native solution conditions that they are difficult to fully remove. In this work, the role of metal ions in biological analytes is explored. For analytes that require metal ions to maintain higher order structures, a mass spectrometry method was developed to determine whether a stable structure is formed from metal ion adducts, or if the metal ion adducts are nonspecifically bound. Electron transfer of these structures reveals complementary fragmentation information, with the added discovery of new radical fragmentation pathways. With mass spectrometry, specific ligand and metal ion affinities can even be determined for analytes at low enough concentrations. In addition to analytes that require metals, an exploration on unwanted metal ion adduction during the electrospray ionization process is shown via gas-phase ion/ion reactions. Observing how specific anionic ligands exchange metals with protons from proteins on a small and controlled scale gives a greater understanding of what solutions can lead to the cleanest results. In addition, this work shows the possibility of finding anionic ligands that will instead exchange protons with metal ions found on proteins. In the gas-phase, these experiments have a high degree of control, leading to a much greater understanding of how metal ions influence mass spectrometry samples.</p>

<p>Microglia are a population of specialized, tissue-resident immune cells that make up around 10% of total cells in our brain. They actively prune neuronal synapses, engulf cellular debris, and misfolded protein aggregates such as the Alzheimer’s Disease (AD)-associated amyloid-beta (Aβ) by the process of phagocytosis. During AD, microglia are unable to phagocytose Aβ, perhaps due to the several disease-associated changes affecting their normal function. Functional molecules such as lipids and metabolites also influence microglial behavior but have primarily remained uncharacterized to date. The overarching question of this work is, <i>How do microglia become dysfunctional in chronic inflammation</i>? To this end, we developed new chemical tools to better understand and investigate the microglial response to Aβ <i>in vitro</i> and <i>in vivo</i>. Specifically, we introduce three new tools. (1) Recombinant human Aβ was developed via a rapid, refined, and robust method for expressing, purifying, and characterizing the protein. (2) A pH-sensitive fluorophore conjugate of Aβ (called Aβ^pH) was developed to identify and separate Aβ-specific phagocytic and non-phagocytic glial cells <i>ex vivo</i> and <i>in vivo</i>. (3) New lysosomal, mitochondrial, and nuclei-targeting pH-activable fluorescent probes (called LysoShine, MitoShine, and NucShine, respectively) to visualize subcellular organelles in live microglia. Next, we asked, <i>What changes occur to the global lipid and metabolite profiles of microglia in the presence of Aβ in vitro and in vivo</i>? We screened 1500 lipids comprising 10 lipid classes and 700 metabolites in microglia exposed to Aβ. We found significant changes in specific lipid classes with acute and prolonged Aβ exposure. We also identified a lipid-related protein that was differentially regulated due to Aβ <i>in vivo</i>. This new lipid reprogramming mechanism “turned on” in the presence of cellular stress was also present in microglia in the brains of the 5xFAD mouse model, suggesting a generic response to inflammation and toxicity. It is well known that activated microglia induce reactive astrocytes during inflammation. Therefore, we asked, <i>What changes in proteins, lipids, and metabolites occur in astrocytes due to their reactive state? </i>We provide a comprehensive characterization of reactive astrocytes comprising 3660 proteins, 1500 lipids, and 700 metabolites. These microglia and astrocytes datasets will be available to the scientific community as a web application. We propose a final model wherein the molecules secreted by reactive astrocytes may also induce lipid-related changes to the microglial cell state in inflammation. In conclusion, this thesis highlights chemical neuroimmunology as the new frontier of neuroscience propelled by the development of new chemical tools and techniques to characterize glial cell states and function in neurodegeneration.</p>

<div>Vibrational spectroscopic imaging has become an emerging platform for chemical visualization of biomolecules and materials in complex systems. For over a century, both Raman and infrared spectroscopy have demonstrated the capability to recognize molecules of interest by harnessing the characteristic features from molecular fingerprints. With the recent development of hyperspectral vibrational spectroscopy imaging, which records the chemical information without sacrificing the spatial-temporal resolution, numerous discoveries has been achieved in the field of molecular and cellular biology. Despite the ability to provide complimentary chemical information to Raman-based approaches, infrared spectroscopy has not been extensively applied in routine studies due to several fundamental limitations: 1). the poor spatial resolution; 2). inevitable strong water absorption; 3). lack of depth resolution.</div><div>Mid-infrared photothermal (MIP) microscopy overcame all the above mentioned problems and for the first time, enabled depth-resolved in vivo infrared imaging of live cells, microorganisms with submicrometer spatial resolution. The development of epi-detected MIP microscopy further extends its application in pharmaceutical and materials sciences. With the deployment of difference frequency generation and other nonlinear optical techniques, the spectral coverage of the MIP microscopy was significantly enhanced to enable chemical differentiation in complex systems across the broad mid-infrared region. In addition to the efforts to directly improve the performance of MIP microscopy, a novel quantitative phase imaging approach based on polarization wavefront shaping via custom-designed micro-retarder arrays was developed to take advantage of the highly sensitive phase measurement in combination with the photothermal effect. Besides, the extended depth-of-field and multifocus imaging enabled by polarization wavefront shaping could both improve the performance of MIP microscopy for volumetric imaging.</div>