The aim of my PhD research was to develop and establish techniques for surface functionalization of nanoparticles, which can be employed to study the dynamics, function and activity of recombinantly expressed as well as endogenous proteins inside living cells. A prerequisite to achieve this goal was the ability to bio-functionalize nanoparticles with proteins in the cytoplasm of living cells. The HaloTag technology was utilized for generic site-specific targeting of nanoparticles to proteins. Fast and efficient targeting of nanoparticles to proteins was then achieved by using an engineered clickHTL exhibiting fast reactivity towards the HaloTag-enzyme. Application of this approach to track individual proteins in the outer membrane of mitochondria revealed that the physicochemical properties of the nanoparticles biased the mobility of the targeted proteins. To circumvent this, a model nanoparticle was systematically engineered in order to identify physicochemical properties that are important for tracking intracellular membrane proteins without affecting their diffusion dynamics. Nanoparticles exhibiting stealth properties were finally obtained upon densely coating the nanoparticle surface with PEG2k. These particles were mono-functionalization with clickHTL, to ensure labeling in a 1:1 stoichiometry, and could be successfully used for unbiased tracking of individual membrane proteins. Beyond the observation of proteins, generic approaches that allow intracellular manipulation and probing of protein activities are desired. To this end, 500 nm superparamagnetic nanoparticles were used as mobile nanoscopic hotspots self-assebled into active signaling platforms. Inside living cells, precise and accurate manipulation of endogeneous Rac1 activity was possible at different subcellular locations and over extended time periods. These experiments demonstrated that Rac1 signaling is dependent on the subcellular-context by spatial isolation of distinct signaling pathways. Furthermore, these MNPs provided well defined platforms for selective spectroscopy in order to quantify bait-prey protein interactions in the cytoplasm as was demonstrated by the interaction of cdc42 and N-WASP.

The global spread of COIVD-19, as well as the worsening air pollution throughout the world have brought tremendous attention into the development of materials that can efficiently capture particulate matter (PM). Traditional filters made from fabric, glass fibers, or melt blown fibers exhibit a low efficiency at removing sub-micrometer and nanoscale particles. Additionally, they exhibit limited performance in high humidity, high temperature environments. We suggest that the high porosity of filters composed of nanofibers could provide minimal obstruction to air flow, while their high tortuosity and surface area-to-volume ratio presents an excellent platform for particle capture. Electrospinning is a simple and well-studied process to produce randomly accumulated nano- and micro-scale diameter fibers. The main advantages of electrospun nanofibers include their tunable fiber morphology and diameter under specific electrospinning parameters, as well as the ease of post-process modification. Studies have demonstrated its promising applications ranging from tissue engineering, water purification to wearable electronics. Giving the tunable aspect of the process, various polymers were electrospun with different morphology and fiber diameter which all demonstrated high particle removal efficiency. Cellulose was chosen as the base material for our study since it is the most abundant biopolymer and its affinity for further chemical modification. In this study, the removal of nanoscale particles via in-house fabricated cellulose nanofilters is significantly enhanced by chemically functionalizing the fibers’ surface via the deposition of the bio-inspired glue polydopamine (PDA) and the polycation poly(diallyldimethylammonium chloride) (PDADMAC). Nanofilters were electrospun from cellulose acetate solutions before being regenerated to cellulose via an alkaline treatment. Cellulose nanofilters were then functionalized using only PDA or the codeposition of PDA with PDADMAC. Scanning electron microscope (SEM), Fourier transform infrared spectrometer (FTIR), and high-resolution X-ray photoelectron spectroscopy (XPS) were used to characterize the nanofilters. The effects of filter packing density, filter layering, and surface functionalization on their performance, i.e., their filtration efficiency, most penetrating particle size (MPPS), performance in a high humidity environment, and filter pressure drop were investigated. Furthermore, by introducing hydrophilic and hydrophobic nanofibers within a composite filter structure, the performance of the composite filter remained unchanged even in high humidity.

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Both the thermal and copper(I) catalyzed azide-alkyne Huisgen cycloadditions were explored as strategies to be used for silicone functionalization and crosslinking. The generality of these reactions was demonstrated (Chapter 2) using 1,3-bis(azidopropyl)tetramethyldisiloxane (BAPTMDS) as a model compound. The ligation of this compound with several alkyne-containing molecules, especially the copper(I) catalyzed process or “click” proved to be easy to perform, high yielding, and gave the 1,4-triazole regioisomer as the sole product. Thermal, metal catalyst-free, azide-alkyne cross-linking (Chapter 3) using a poly(azidopropylmethylsiloxane)-co-dimethylsiloxane as the base polymer and several polysubstituted alkyne molecules as crosslinkers was efficient. The reaction of the base polymer with an ethynyl-terminated disiloxane demonstrates that a silicone elastomer can be synthesized by simple heating and that the resulting material is stable, decomposing only at temperatures higher than 230 °C. Finally, direct bioconjugation of silicones to biotin using propargylamide and BAPTMDS was examined (Chapter 4). The result of the copper(I) catalyzed Huisgen ligation of biotin onto silicones was as efficient as the reactions in the previous chapters, revealing that the “click” process can successfully be applied to a broad range of silicones. / Thesis / Master of Science (MSc)

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Single-w ailed carbon nanotubes (SWNTs) were oxidatively shortened and functionalized with ruthenium-based olefin metathesis catalysts. These catalyst-functionalized nanotubes were shown to be effective in the ring-opening metathesis polymerization of norbornene, resulting in rapid polymerization from the catalyst sites on the nanotube. It was found that high polymer molecular weights could be reached, and the molecular weight increased linearly with polymerization time. The resulting polynorbomene-functionalized nanotubes were found to exhibit solubility in organic solvents, while the starting materials and catalyst-functionalized nanotubes were completely insoluble. The polymerized materials were characterized by NMR, IR, DSC, AFM and TEM. Polystyrene and poly[(t-butyl acrylate)-b-styrene] with well-defined molecular weights and polydispersities were prepared by nitroxide-mediated free-radical polymerization. The homo- and block-copolymers were used to functionalize shortened single-walled carbon nanotubes (SWNTs) through a radical coupling reaction involving polymer-centered radicals generated at 125°C via loss of the stable free-radical nitroxide capping agent. The resulting polymer-SWNT composites were fully characterized and were found to be highly soluble in a variety of organic solvents. This solubility could also be altered through chemical modification of the appended polymers. The t-butyl groups of appended PtBA-b-PS could be removed to produce poly[(acrylic acid)-b-styrene]- functionalized carbon nanotubes. The resulting composite was found to form aggregates in a mixture of chloroform/methanol (v/v: 1/1), as determined by dynamic light scattering (DLS). / Thesis / Master of Science (MS)

Multiple hurdles, such as drug solubility, stability, and physical barriers in the body, hinder bioavailability of many promising therapeutics. Polymeric nanocarriers can encapsulate the therapeutics to protect non-target areas from side effects but also protect the drug from premature degradation for increased circulation and bioavailability. To capitalize on these advantages, the polymer nanoparticle must be properly engineered for increased control in size distribution, therapeutic encapsulation, colloidal stability, and release kinetics. However, each application requires a specific set of characteristics and properties. Being able to tailor these by manipulation of different design parameters is essential to optimize nanoparticles for the application of interest. This study of nanoparticle fabrication and characterization takes us a step closer to building effective delivery systems tailored for specific treatments. Poly(ethylene oxide)-b-poly(D,L-lactic acid) (PEO-b-PDLLA) based nanoparticles were produced to range from 100-200 nm in size. They were fluorescently labeled with a hydrophobic dye 6-13 bis(triisopropylsilylethynyl) pentacene (TIPS pentacene) at an optimal loading of 0.5 wt% with respect to the core. Surfaces were successfully coated with streptavidin to be readily functionalized with various biotinylated compounds such as PD-L1 antibodies or A488 fluorophore. Using the same PEO-b-PDLLA, iron oxide and a conjugated polymer poly(2- methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene) (MEH-PPV) were co-encapsulated to form fluorescently labeled magnetic particles. Using poly(lactic-co-glycolic acid), CRISPR-Cas9 plasmids were encapsulated at 1.6 wt% and most of the payload released within the first 24 hours. The incorporated plasmids were intact enough to have mammalian macrophages successfully express the bacterial protein Cas9. Using similar PLGA based particles, the surface was functionalized with streptavidin and bound to the surface of bacteria as an active carrier for increased penetration of solid tumors averaging ~23 particles per bacterium. PEO-b-PLGA based particles were used in conjunction with a hydrophobic salt former to encapsulate a peptide designed to reduce platelet binding to cancer cells and mitigate extravasation. The peptide encapsulated was increased from < 2 wt% without salt former to 8.5 wt% with the used of hexadecyl phosphonic acid. Although the applications across these projects can be broad, the fundamentals and important design parameters considered contribute to the overarching field of effective carriers for drug delivery. / Ph. D. / There are many reasons why many promising pharmaceutical formulations never make it through regulation and onto market, including low solubility of the drug, low absorbance by the body, and harmful side effects, to name a few. Using polymer drug carriers, these difficulties can be overcome by holding the drug in a more soluble carrier, releasing it on a certain timeline or to a specific location to increase absorbance and decrease side effects. When designing a carrier, the requirements for the product are dependent on the application and the disease of interest. This work looks at the material types and conditions during particle formation to see how it affects the final product to better define and understand how these parameters change the performance. This work shows that the carrier size can be manipulated depending on how much of one material is used versus the other, they can be labeled to fluoresce so they can be tracked during cell and animal studies, and they can be coated with targeting compounds on the surface to increase the specificity of the carrier to localize to a target location of interest. Different particles containing DNA for gene editing, peptides for cancer therapies, and magnetic iron oxides to increase transport across difficult cell barriers have all be fabricated and characterized. The lessons learned through these projects will help guide future work to more effective and efficient delivery of pharmaceuticals to the body.

Nanographene oxide particles (NGO) were produced via oxidative exfoliation of graphite. Three different sizes of NGO (300 nm, 200 nm and 100 nm) have been separated by using probe sonication and sucrose density gradient centrifugation. There is great interest in functionalized NGO as a nanocarrier for in vitro and in vivo drug delivery, in order to improve dispersibility and stability of the nanocarrier platforms in physiological media. In this study, the NGO particles were covalently functionalized with zero generation polyamidoamide (PAMAM-G0) and with gelatin via noncovalent interaction. Spectroscopic techniques have been used to discriminate the chemical states of NGO prior and after functionalization. The X-ray photoelectron spectroscopy (XPS) revealed a clear change in the chemical state of NGO after functionalization, for both covalent and noncovalent approaches. Raman spectroscopy gave obvious insight after oxidation of graphite and functionalization of NGO particles depending on the variation of intensity ratios between D, G and 2D bands. The Fourier transform infrared spectroscopy (FTIR) exhibited the presence of oxygen containing functional groups distributed onto graphene sheets after oxidation of graphite. Furthermore, the FTIR is complementary with the XPS which performed a strong reduction in the oxygen contents after functionalization. UV visible spectroscopy was used to understand the binding capacity of gelatin coated NGO particles. The Microscopy tools, scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used to estimate the dimensions of NGO particles (thickness and lateral width). The nanohybrid systems (NGO-PAMAM and Gelatin-NGO) loaded with carboplatin (CP) were sought for anticancer activity investigation in HeLa and neuroblastoma cancer cells respectively. Mesenchymal stem cells (hMSCs) were used as a model of normal cells. On HeLa cells, the pristine NGO particles with average widths of 200 nm and 300 nm showed a cytotoxic effect at low (50 g.ml−1) and high (100 g.ml−1) concentrations. While the pristine NGO sample with an average width of 100 nm revealed no significant cytotoxicity at 50 g.ml−1, and only recorded a 10% level at 100 g.ml−1. The mesenchymal stem cells showed less than 35% viability for all size distributions. After functionalization with PAMAM, the carrier was found to be able to deliver carboplatin to the cancer cells, by enhancing the drug anticancer efficiency. Moreover, the carboplatin loaded NGO carrier shows no significant effect on the viability of hMSCs even at high concentration (100 g.ml−1). On neuroblastoma cells, the cell viability assay validated gelatin-NGO nanohybrids as a useful nanocarrier for CP release and delivery, without obvious signs of toxicity. The nano-sized NGO (200 nm and 300 nm) did not enable CP to kill the cancer cells efficiently, whilst the CP loaded gelatin-NGO 100 nm resulted in a synergistic activity through increasing the local concentration of CP inside the cancer cells.

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