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The growth of the multibillion dollar bionanoparticle industry has spurred the development of new physical characterization methods. One such method, electrospraydifferential mobility analysis (ES-DMA) constitutes an electrospray for aerosolization of bionanoparticles (such as viruses, gold-nanoparticles, proteins, nanoparticle-protein complexes) and an ion mobility method that operates at atmospheric conditions, and separates bionanoparticles spatially. This dissertation identifies some relevant "problem" areas for ES-DMA by reviewing selected applications.Some such problems are: proteins while passing through ES capillaries are found to interact with it and thus produce time dependent size distributions. Further, it is thought that adsorbed proteins may subsequently desorb and influence size distributions with the ES-DMA which may concomitantly affect quantification of aggregates. These artifacts are studied systematically and it is demonstrated that ES-DMA can quantify adsorption-desorption of complex protein mixtures at high shear rates. Further, it is shown that desorbing proteins do not have a significant effect on size distributions. Another artifact of the ES takes place during the aersolization process. Two units (called monomers) of a bionanoparticle may get encapsulated in the same ES droplet and upon drying of the droplet create artificial dimers thus affecting quantification with ES-DMA. Assuming Poisson distribution, this thesis provides a systematic approach that can be undertaken to eliminate this artifact. A third artifact arises from the low sensitivity of the DMA to size increase. When a ligand (for e.g. protein) adsorbs to a bionanoparticle it creates an increase in the size of the later, which can be used to quantify the amount of ligand adsorbed per bionanoparticle. As ligands can change conformations upon adsorption, using ES-DMA for such applications may be flawed. This issue has been identified and a solution has been provided by integrating a mass analyzer after the ES-DMA.After correcting for these artifacts, this dissertation delves into characterization of different types of bionanoparticles and demonstrates that ES-DMA has several advantages over other traditional techniques such as transmission electron microscopy, size exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and plaque assay and thus has immense potential to become a process analytical technique in biomanufacturing environments. ELECTROSPRAY-DIFFERENTIAL MOBILITY ANALYSIS OF BIONANOPARTICLES

Electrospray (ES) sources are commonly used to introduce nonvolatile materials (e.g., nanoparticles, proteins, etc.) to the gas phase for characterization by mass spectrometry or ion mobility. Recent studies in our group using ES ion mobility to characterize protein aggregation in solution have raised the question as to whether the ES itself induces aggregation and thus corrupts the results. In this article, we develop a statistical model to determine the extent to which the ES process induces the formation of dimers and higher-order aggregates. The model is validated through ES differential mobility experiments using gold nanoparticles. The results show that the extent of droplet-induced aggregation is quite severe and previously reported cutoff criterion is inadequate. We use the model in conjunction with experiment to show the true dimer concentration in a protein solution as a function of concentration. The model is extendable to any ES source analytical system and to higher aggregation states. For users only interested in implementation of the theory, we provide a section that summarizes the relevant formulas.

The mobility of a nonspherical particle is a function of both particle shape and orientation. Thus, unlike spherical particles, the mobility, through its orientation, depends on the magnitude of the electric field. In this work, we develop a general theory, based on an extension of the work of Happel and Brenner (1965), for the orientation-averaged mobility applicable to any axially symmetric particle for which the friction tensor and the polarization energy are known. By using a Boltzmann probability distribution for the orientation, we employ a tensor formulation for computing the orientation-averaged mobility rather than a scalar analysis previously employed by Kim et al. (2007) for nanowires. The resulting equation for the average electrical mobility is much simpler than the expression based on the scalar approach, and can be applied to any axially symmetric structures such as rods, ellipsoids, and touching spheres. The theory is applied to the specific case of nanowires and the experimental results on the mobility of carbon nanotubes (CNT). A set of working formulas of additional mobility expressions for nanorods and prolate spheroids in the free molecular, continuum, and transition regimes are also presented. Finally, we examine the expression of dynamic shape factor common in the literature, and propose a clearer definition based on the tensor approach. Mathematica codes for the electrical mobility evaluations for five cases are provided in the Supplemental Information.

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We have developed a simple, fast, and accurate method to measure the absolute number concentration of nanoparticles in solution. The method combines electrospray differential mobility analysis (ES-DMA) with a statistical analysis of droplet-induced oligomer formation. A key feature of the method is that it allows determination of the absolute number concentration of particles by knowing only the droplet size generated from a particular ES source, thereby eliminating the need for sample-specific calibration standards or detailed analysis of transport losses. The approach was validated by comparing the total number concentration of monodispersed Au nanoparticles determined by ES-DMA with UV/vis measurements. We also show that this approach is valid for protein molecules by quantifying the absolute number concentration of Rituxan monoclonal antibody in solution. The methodology is applicable for quantification of any electrospray process coupled to an analytical tool that can distinguish monomers from higher order oligomers. The only requirement is that the droplet size distribution be evaluated. For users only interested in implementation of the theory, we provide a section that summarizes the relevant formulas. This method eliminates the need for sample-specific calibration standards or detailed analysis of transport losses.

The mobility of a nonspherical particle is a function of both particle shape and orientation. In turn, the higher magnitude of electric field causes nonspherical particles to align more along the field direction, increasing their mobility or decreasing their mobility diameter. In previous works, Li et al. developed a general theory for the orientation-averaged mobility and the dynamic shape factor applicable to any axially symmetric particles in an electric field, and applied it to the specific cases of nanowires and doublets of spheres. In this work, the theory for a nanowire is compared with experimental results of gold nanorods with known shape determined by TEM images. We compare the experimental measured mobility sizes with the theoretical predicted mobility in the continuum, free molecular, and the transition regime. The mobility size shift trends in the electric fields based on our model, expressed both in the free molecular regime and in the transition regime, are in good agreement with the experimental results. For rods of dimension: width d r = 17 nm and length L r = 270 nm, where one length scale is smaller than the mean free path and one larger, the results clearly show that the flow regime of a slender rod is mostly controlled by the diameter of the rod (i.e., the smallest dimension). In this case, the free molecule transport properties best represented our nanorod. Combining both theory and experiment we show how, by evaluating the mobility as a function of applied electric field, we can extract both rod length and diameter.

There is an urgent need to design and develop new and more potent EGFR inhibitors with improved anti-tumor activity. Here we describe the design and synthesis of two series of 4-benzothienyl amino quinazolines as new analogues of the EGFR inhibitor Gefitinib. The anti-tumor activity of these novel Gefitinib analogues in 6 human cancer cell lines was examined. Compared with the parental Gefitinib, most of the new compounds show a markedly increased cytotoxicity to cancer cells. Furthermore, several of the series B compounds that side chains at position 7 contain either a methyl or ethyl group are potent pan-RTK inhibitors. Two representative compounds in this class, 15 and 17, have an enhanced capability to inhibit cancer cell growth and induce apoptosis in vitro and inhibit tumor formation in vivo in human cancer cells with high HER-2, as compared with the parental Gefitinib. Thus they may be promising lead compounds to be developed as an alternative for current Gefitinib therapy or for Gefitinb-resistant patients, potentially via simultaneously blocking multiple RTK signaling pathways.