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Capturing protein dynamics in biological crowded environments is essential for under- standing cellular function. In this project, we have explored the dynamic properties of apoferritin in aqueous solutions under varying conditions, including different temper- ature, solvent viscosity and protein concentrations. Dynamic light scattering (DLS) was applied here at various scattering angles from 90 to 150 degrees and at temperatures 295 K and 263 K on three different samples, including one with 19.5 mg/ml apo- ferritin, 6 mg/ml NaCl and 50% glycerol in volume fraction, one with 20 mg/ml apoferritin and 6 mg/ml NaCl, and one with 196.7 mg/ml apoferritin, 6 mg/ml NaCl and 50% glycerol in volume fraction. With the intensity autocorrelation func- tions from DLS measurements, the corresponding diffusion coefficients, hydrodynamic radii and relaxation time constants for each sample under varying conditions were ex- tracted. By comparing with the previous studies, unexpectedly large hydrodynamic radii were noticed and were attributed to undissolved protein crystallites. Still, it can be indicated from our experiment that applying smaller momentum transfer, decreas- ing temperature, increasing solvent viscosity and increasing protein concentration in the solutions can slow down the diffusion dynamics of protein molecules and clusters. Especially by increasing protein concentration, the slowing down of dynamics may be due to crowding effects, as well as increased size of the crystallites. In addition, the data indicate that in all cases, larger solution viscosity can lead to slower diffusivity of proteins.

The objective of this research is to further define the relationship between the charge state of insulin, and the self assembly properties of insulin and PEGylated insulin in solution. Polyethylene glycol (PEG) chains were covalently attached to insulin in order to evaluate their impact on insulin¿s systemic duration of action after pulmonary dosing. This thesis will focus on the assembly properties of the PEG-insulin and insulin, and also demonstrate how the charge state, which was modified by the covalent attachment of PEG, relates to different modes of behavior by anion and cation exchange chromatography. In addition, explain how modifying the assembly state extends to improving formulation properties of spray-dried insulin powders. This thesis is an investigation into the relationship of insulin¿s charge state controlled by pH and how the charge state affects the self assembly of insulin, especially when the zinc ion is removed. Ionic interaction is one of the major forces affecting insulin assembly. The theory that a change in the charge state of insulin could modulate the ionic interaction and reduce hexamer formation at alkaline conditions was investigated. Experiments were designed to measure the level of hexamer with light scattering, and the amount of hexamer was then correlated with the pH and zinc content of the solutions. The importance of the charge state of the monomer and its behavior extends to chromatography and purification modes as well. Specifically, the purification of various species of PEGylated insulin presents a challenge. By varying mobile phase pH which induces the charge to insulin, an ion exchange method demonstrated very high resolution and controllable interaction between the ion exchange media and the insulin derivatives. A highly accurate method for determining molecular weight and thus the average associated state of insulin in solution has been developed using the MALS (Multi-Angle Light Scattering). Insulin concentration, pH, and metal ion concentrations, were in pharmaceutically relevant ranges. The MALS method was developed to evaluate how the parameters above affect the self-assembly properties of insulin, and use the assembly properties to improve formulations of insulin or PEGylated insulin. To use the light scattering technique the dn/dc (change in refractive index with change in concentration) is required. During the method development, the dn/dc of insulin was measured at 690 nm, and a value of 0.185 mL/g based on theory was confirmed. A novel approach for preparing insulin powders with improved chemical stability, based on maintaining the dissociation of hexamers in solution during the spray drying process was developed. The mode presented here is to remove the zinc ions from solution, increase the pH from 6.6 to 7.8, and maintain a low concentration of insulin approximately 2 to 15 mg/mL. Each of these factors alone decreases the hexamer population in solution, but by combining all three factors, hexamers are driven to very low levels of equilibrium. The increased stability of the powders is predominately related to the decrease in covalent insulin dimer (CID). The data presented correlates a reduced hexamer population in the solution with lower levels of CID¿s in the dry powder compared to controls. The CID formation rate was reduced by 40% compared to a control.

Liquid-liquid phase separation (LLPS) is an important phenomenon in soft condensed matter that explains many properties of membraneless organelles in living cells. The research on this topic is, therefore, a field with a wide range of applications such as biopharmacy and biomaterials. In this project, we investigate the LLPS of the antibody protein Immunoglobulin G (IgG) by analyzing the liquid dynamics of IgG solutions at a wide range of temperatures with dynamic light scattering (DLS). It was found that the slow component of the autocorrelation function increases with decreasing temperature below 0 °C. This can be attributed to either the number of protein clusters increasing as the sample approaches phase separation or LLPS droplets forming in the solution. LLPS was detected through optical microscopy, visualising the droplet formation in the IgG solution. This work confirms that LLPS can be detected for bovine IgG solutions without the presence of cosolvents and without water freezing in the sample.

The reliance on low-viscosity and photoactive resins limits the accessible properties for vat photopolymerization (VP) materials required for engineering applications. This has limited the adoption of VP for producing end-use parts, which typically require high MW polymers and/or more stable chemical functionality. Decoupling the viscosity and molecular weight relationship for VP resins has been completed recently for polyimides and highperformance elastomers by photocuring a scaffold around polymer precursors or polymer nanoparticles, respectively. Both of these materials are first shaped by printing a green part followed by thermal post-processing to achieve the final part properties. This dissertation focuses on improving the processability of these material systems by (i) investigating the impact of scaffold architecture and polysalt monomer composition on photocuring, thermal post-processing, and resulting thermomechanical properties and (ii) developing a Monte Carlo ray-tracing (MCRT) simulation to predict light scattering and photocuring behavior in particle-filled resins, specifically zinc oxide nanoparticles in a rigid polyester resin and styrene butadiene rubber latex resin. The first portion of the dissertation introduces VP of a tetra-acid and half-ester-based polysalt resin derived from 4,4'-oxydiphthalic anhydride and 4,4-oxydianiline (ODPA-ODA), a fully aromatic polyimide with high glass transition temperature and thermal stability. This polyimide, and polyimides like this, find use in demanding industries such as aerospace, automotive and electronic applications. The author evaluated the hypothesis that a non-bound triethylene glycol dimethacrylate (TEGDMA) scaffold would facilitate more efficient scaffold burnout and thus achieve parts with reduced off-gassing potential at elevated temperatures. Both resins demonstrated photocuring and were able to print solid and complex latticed parts. When thermally processed to 400 oC, only 3% of the TEGDMA scaffold remained within the final parts. The half-ester resin exhibits higher char yield, resulting from partial degradation of the polyimide backbone, potentially caused by lack of solvent retention limiting the imidization conversion. The tetra-acid exhibits a Tg of 260oC, while the half-ester displays a higher Tg of 380 oC caused by the degradation of the polymer backbone, forming residual char, restricting chain mobility. Solid parts displayed a phase-separated morphology while the half-ester latticed parts appear solid, indicating solvent removal occurs faster in the half-ester composition, presumably due to reduced polar acid functionality. This platform and scaffold architecture enables a modular approach to produce novel and easily customizable UV-curable polyimides to easily increase the variety of polyimides and the accessible properties of printed polyimides through VP. The second section of this dissertation describes the creation and validation of a MCRT simulation to predict light scattering and the resulting photocured shape of a ZnO-filled resin nanocomposite. Relative to prior MCRT simulations in the literature, this approach requires only simple, easily acquired inputs gathered from dynamic light scattering, refractometry, UV-vis spectroscopy, beam profilometry, and VP working curves to produce 2D exposure distributions. The concentration of 20 nm ZnO varied from 1 to 5 vol% and was exposed to a 7X7 pixel square ( 250 µm) from 5 to 11 s. Compared to experimentally produced cure profiles, the MCRT simulation is shown to predict cure depth within 10% (15 µm) and cure widths within 30% (20 µm), below the controllable resolution of the printer. Despite this success, this study was limited to small particles and low loadings to avoid polycrystalline particles and maintain dispersion stability for the duration of the experiments. Expanding the MCRT simulation to latex-based resins which are comprised of polymer nanoparticles that are amorphous, homogeneous, and colloidally stable. This allows for validating the MCRT with larger particles (100 nm) at higher loadings. Simulated cure profiles of styrene-butadiene rubber (SBR) loadings from 5 vol% to 25 vol% predicted cure depths within 20% ( µm) and cure widths within 50% ( µm) of experimental values. The error observed within the latex-based resin is significantly higher than in the ZnO resin and potentially caused by the green part shrinking due to evaporation of the resin's water, which leads to errors when trying to experimentally measure the cure profiles. This dissertation demonstrates the development of novel and functional materials and creation process-related improvements. Specifically, this dissertation presents a materials platform for the future development of unique photocurable engineering polymers and a corresponding physics-based model to aid in processing. / Doctor of Philosophy / Vat Photopolymerization (VP) is a 3D printing process that uses ultraviolet (UV) light to selectively cure liquid photosensitive resin into a solid part in a layer-by-layer fashion. Parts produced with VP exhibit a smooth surface finish and fine features of less than 100 µm (i.e., width of human hair). Recoating the liquid resin for each layer limits VP to low-viscosity resins, thus limiting the molecular weight (and thus performance) of the printed polymers accessible. Materials that are low molecular weight are limited in achieving desirable properties, such as elongation, strength, and heat resistance. Solvent-based resins, such as polysalt and latex resins have demonstrated the ability to decouple the viscosity and molecular weight relationship by eliminating polymer entanglements using low-molecular-weight precursors or isolating high-molecular-weight polymers into particles. This dissertation focuses on expanding and improving the printability of these methods. The second chapter of the dissertation investigates the impact of scaffold architecture in printing polyimide polysalts to improve scaffold burnout. Polysalts are polymers that exist as dissolved salts in solution, with each monomer holding two electronic charges. When heated, the solvent evaporates and the monomers react to form a high molecular-weight polymer. While previous work featured a polysalt that was covalently bonded to the monomers, the polysalt in this work is made printable by co-dissolving a scaffold. The polysalt resins are photocured and thermally processed to polymerize and imidize into a high-molecular-weight polymer, while simultaneously pyrolyzing the scaffold. Using a co-dissolved scaffold allows the investigation of two different monomers of tetra-acid and half-ester functionality. The half-ester composition underwent degradation during heating, increasing the printed parts' glass transition or softening point. The scaffold had little impact on the polysalt polymerization or final part properties and was efficiently removed, with only 3% remaining in final parts. The composition and properties of the monomers selected played a bigger role due to partial degradation altering the properties of the final parts. Overall, this platform and scaffold architecture allows for a larger number of polyimides to be accessible and easily customizable for future VP demands. The third chapter describes the challenges of processing photocurable resins that contain particles due to the UV light scattering in the resin vat during printing. When the light from the printer hits a particle, it is scattered in all directions causing the layer shape to be distorted from the designed shape. To overcome this, a Monte Carlo ray-tracing (MCRT) simulation was developed to mimic light rays scattering within the resin vat. The simulation was validated by comparing simulation results against experiment trials of photocuring resins containing 20nm zinc oxide (ZnO) nanoparticles. The MCRT simulation predicted all the experimental cure depths within 10% (20 µm) and cured widths within 30% (15 µm) error. Despite the high accuracy, this study was limited to small particles and low concentrations. Simulating larger particles is difficult as the simulation assumes each particle to be uniform throughout its volume, which is atypical of large ceramic particles. The fourth chapter enables high particle volume loading by using a highly stretchable styrene-butadiene rubber (SBR) latex-based resin. Latex-based resins maintain low viscosity by separating large polymer chains into nano-particles that are noncrystalline and uniform. When the chains are separated, they cannot interact or entangle, keeping the viscosity low even at high concentrations (>30 vol%). Like the ZnO-filled resin, the latex resin is experimentally cured and the MCRT simulation predicts the resulting cure shape. The MCRT simulation predicted cure depths within 20% (100 µm) and over-cure widths within 50% (100 µm) of experimental values. This error is substantially higher than the ZnO work and is believed to be caused by the water evaporating from the cured resin resulting in inconsistent measurements of the cured dimensions.