Title: Self-assembly of polyelectrolytes in aqueous solutions (dissipative particle dynamics) Author: Karel Šindelka Department: Faculty of science, Charles University in Prague Supervisor: Doc. Ing. Zuzana Limpouchová, CSc. Consultant: Doc. Ing. Martin Lísal, DSc. Abstract In the thesis, the coarse-grained dissipative particle dynamics (DPD) is used for the study of self-assembly of equimolar mixtures of oppositely charged symmetric block PEs with one PE block (either strong polycation or strong polyanion) and one readily water-soluble neutral block in aqueous media. In the first part of the diploma thesis, the principles of DPD simulations are described and the correct implementation of electrostatic interactions in the DPD method is demonstrated on the example of counterion (Manning) condensation. In the second main part, the effect of the blocks solubility, incompatibility and the interplay of different forces on electrostatic assembly is investigated. The cor- responding neutral systems are also simulated for comparison. The study shows that the hydrophobicity of the PE backbone and the incompatibility of blocks sig- nificantly affects the electrsotatic co-assembly. The presence of opposite charges on different chains promotes the aggregation process and the aggregation number in- creases in comparison...

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A polyelectrolyte complex is formed by mixing two oppositely charged polyelectrolytes in a solution. The electrostatic interactions between partially charged polymeric chains lead to the formation of a stable complex while avoiding the use of covalent cross linkers. Since complex formation can improve the stability of polyelectrolyte and metal ions in polyelectrolyte can provide various functionalities, PECs incorporated with metal ions are promising candidates for manufacturing stable and multifunctional structures. While the coordination of metal ions and polyelectrolytes has been extensively investigated in solutions and multilayer films, to our knowledge, no research has been performed to study the effect of metal ion/polyelectrolyte interactions on PECs structures and properties. The following research demonstrates the impact of different metal ions in controlling PEC structure morphology and applications. These discoveries indicate great potential of metal ions in PECs to fabricate functional PEC nanostructures. The research investigates the effect of the interactions between different metal ions and polyelectrolytes on the morphology and properties of PECs, explore the fabrication of different structures using embedded metal ions and understand the impact of metal ion/polyelectrolyte interactions on the nanoparticle structures. The research concludes: 1) incorporating metal ions of different valence into PECs introduces metal ion/polyelectrolyte interactions that can tune the morphology of PECs; 2) metal ion/polyelectrolyte interactions can be used to control the PECs swelling properties and stability in aqueous solutions; 3) the release of embedded metal ions from PECs to aqueous solutions is affected by metal ion/polyelectrolyte interactions; and 4) the embedded metal ions function as a reagent reservoir for various applications to produce functional structures.

<p> The effect of low temperature on the physico-chemical treatment (PCT) of domestic wastewater is examined with special emphasis placed on activated carbon adsorption. PCT is a recent processing scheme, wherein wastewater goes through sedimentation, precipitation, deep bed filtration and adsorption. In PCT, activated carbon adsorption takes the place of conventional biological treatment for organic removal.</p> <p> Both batch studies using powdered activated carbon (PAC) and continuous flow studies with granular activated carbon (GAC) on a pure compound, sodium dodecyl sulfate (SDS) and domestic wastewater from the Dundas, Ontario Water Pollution Control Plant were investigated.</p> <p> Theoretical analysis and batch feasibility studies indicate that the effect of low temperature on coagulation, flocculation and sedimentation were quite easily handled within established design parameters. Batch studies on PAC treatment show that the carbon floc is easily separable from wastewater with the aid of a small dosage of polyelectrolyte and high effluent quality is obtainable at temperatures ranging from 2° - 25°C.</p> <p> Granular activated carbon was evaluated first on a pure compound, SDS. Batch isotherms and kinetics were also conducted at the same time to determine parameters needed for continuous flow modelling. Results derived from this phase of the study indicate that the activation energy from column studies is quite low (approximately 3.0 kcal/mole). Close match is obtained between the theoretical model (a modification of Thomas' reaction kinetics) and experimental data.</p> <p> Finally PCT pilot plant studies on domestic wastewater were carried out, at 5°C and 25°C. Soluble organic carbon removal was in excess of that predicted by purely adsorption type phenomenon. This is attributed to microbial activity inside the carbon columns. Greater soluble organic removal was seen at 25°C than at 5°C. Denitrification inside the carbon columns was found to be quite significant, again with stronger denitrifying activity at 25°C than at 5°C. Microbiological investigation of the carbon from the exhausted columns revealed over 10^9 cells/cm^3) of GAC bed volume from the lead column at 25°C. Denitrifiers were also detected in the carbon. Iodine numbers were determined on used carbon, which indicated the exhaustion of activated carbon capacity. Organic removal, however, continued undiminished, due to bacterial activity.</p> <p> The study culminated with the development of a theoretical model incorporating bacterial activity. The results of column effluent profile, however, indicated great discrepancy between theoretical predictions and experimental observations. This was attributed to simplistic assumptions utilized in solving the fixed bed problem.</p> / Thesis / Master of Engineering (MEngr)

Polyelectrolyte complex coacervates (PECCs) result from liquid-liquid phase separation (LLPS) in solutions containing oppositely charged polymers 1. Multiphase polyelectrolyte complex coacervates (MPECCs) result from the combination of multiple, specific PECCs 2. The encapsulation of proteins in PECCs can serve as promising vehicles for the effective delivery of protein-based therapeutics, which are notoriously difficult to deliver. The encapsulation of model proteins, such as Bovine Serum Albumin (BSA) 3 or Human Hemoglobin (Hb) 4 have illustrated the protein-encapsulating capabilities of these PECC systems. The encapsulation of proteins in MPECCs is a topic that has yet to be explored; however, it can serve to mimic the structure and function of multiphase membraneless organelles, which are abundantly available in cells. This project sought to understand and quantify the encapsulation of enzymes in both PECC and MPECC models; as well as evaluate their efficiency upon encapsulation, as enzymes are simply proteins with catalytic functions 5. A synthesized library of charged, heterochiral polypeptides were used to form both PECC and MPECC systems. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were the enzymes chosen to be assessed in both PECC and MPECC systems. Turbidity measurements, in terms of percent mole of polycation, were used to determine the optimal stoichiometric ratio between the polyanion and polyanion, in the presence of a given concentration of both or either enzyme, in which maximum complex formation occurred. Here we report that a 1:1 stoichiometric ratio of polycation to polyanion in either a solution with 25ug/mL HRP and 25ug/mL GOx, a solution with 50ug/mL GOx, or a solution with 50ug/mL HRP leads to the highest level of complexation. Enzyme encapsulation efficiency of individual PECCs for both enzymes was assessed using the Bradford assay, in which the supernatant was used to determine the concentration of enzyme left in the PECC post-centrifugation. Here we report that all PECC systems were able to encapsulate both GOx and HRP. Higher encapsulation efficiencies were seen with GOx samples compared to HRP samples. Enzymatic activity and efficiency were assessed using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay in the presence of ß-D-glucose. The chromogenic change in intensity over time of each sample was assessed using optical microscopy. Michaelis-Menten graphs were made from the data collected. The resulting data was used to evaluate the Km and Vmax of the enzyme cascade in PECC and MPECC systems compared to a control. Here we report that enzyme cascade efficiency varied among PECC and MPECC samples, with some being more efficient than others. We find that both PECC and MPECC systems generally have lower enzyme-substrate affinity (higher Km) compared to performing the reactions in water. However, this may be related to the need for the substrate to diffuse into a different phase or phases. Interestingly, many of the PECC and MPECC systems have lower Vmax values compared to the water control, indicating a faster enzyme saturation. The enzyme kinetics and efficiency could also be controlled by varying the location of the enzymes in each phase within the MPECC systems. Overall, we show that using MPECC systems allows one to select advantageous properties of individual PECCs and combine them together.

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Polyelectrolyte complexes (PECs) between chitosan, a mucoadhesive, intestinal mucosal permeability-enhancing polysaccharide, and cellulose nanocrystals, rod-like cellulose nanoparticles with sulfate groups on their surface, have potential applications in oral drug delivery. The purpose of this research was to develop an understanding of the formation and properties of chitosan–cellulose nanocrystal PECs and determine their in vitro drug release properties, using caffeine and ibuprofen as model drugs. Cellulose nanocrystals were prepared by sulfuric acid hydrolysis of bleached wood pulp. Chitosans with three different molecular weights (81, 3·103, 6·103 kDa) and four different degrees of deacetylation (77, 80, 85, 89%) were used. PEC formation was studied by turbidimetric titration. PEC particles were characterized by FT-IR spectroscopy, scanning electron microscopy, dynamic light scattering, and laser Doppler electrophoresis. The formation and properties of chitosan–cellulose nanocrystal PEC particles were governed by the strong mismatch in the densities of the ionizable groups. The particles were composed primarily of cellulose nanocrystals. Particle shape and size strongly depended on the mixing ratio and pH of the surrounding medium. The ionic strength of the surrounding medium, and the molecular weight and degree of deacetylation of chitosan had a minor effect. Release of caffeine from the chitosan–cellulose nanocrystal PEC particles was rapid and uncontrolled. Ibuprofen-loaded PEC particles showed no release in simulated gastric fluid and rapid release in simulated intestinal fluid. Further evaluation studies should focus on the expected mucoadhesive and permeability-enhancing properties of chitosan–cellulose nanocrystal PEC particles. / Ph. D.

A major challenge in hepatic tissue engineering is that liver cells rapidly lose their phenotype in in vitro cell culture systems. For this reason, it is necessary to design biomaterials that can support and enhance hepatic functions. Hepatocytes have a surface protein, called the asialoglycoprotein receptor (ASGP-R), which interacts with galactose via a specific receptor-ligand interaction. Polyelectrolyte multilayers (PEMs) were prepared by the layer by layer method, which is based on electrostatic attractions between oppositely charged polyelectrolytes (PEs). Anionic (hyaluronic acid) and cationic (chitosan and galactosylated chitosan) PEs were used in the fabrication of detachable, free-standing PEMs. The main focus of this study is the design of PEMs comprised of 50 bilayers of PEs. PEMs that contained galactose functional groups were assembled with either 5 or 10 bilayers of galactosylated chitosan (5 - 10 % of galactosylation). Optical properties, solvent stability and surface topography of the PEMs were measured. / Master of Science

Wide literature precedence exists for polymers containing electrostatic interactions and polymers containing hydrogen bonding motifs, however the combination of electrostatic and hydrogen bonding interactions is not widely investigated in current literature. Polyelectrolytes containing hydrogen bonding groups are expected to exhibit properties of both classes of supramolecular interactions. A series of adenine- and thyminecontaining PDMAEMA and tert-butyl acrylate copolymers were synthesized to investigate the effect of incorporating hydrogen bonding groups into a polyelectrolyte. Incorporation of the styrenic nucleobases significantly affected the solubility of these copolymers on aqueous solutions and showed salt-triggerability with higher contents of these groups. Polyelectrolytes are capable of binding and condensing DNA through electrostatic interactions with the negatively charged phosphate groups of the DNA backbone; however a high degree of cytotoxicity is also often observed for these gene delivery systems. The high level of cytotoxicity is attributed to high degree of cationic character for the polyplexes formed with these systems according to the proton-sponge hypothesis. One method of reducing the overall cationic character for these systems is incorporation of non-electrostatic binding mechanisms such as hydrogen bonding. A series of nucleobase-containing PDMAEMA copolymers were utilized in order to investigate the effect of incorporation of these groups on the cell viability, binding efficiency, and transfection efficiency of PDMAEMA. / Master of Science

Tobacco has been studied as a host for producing recombinant therapeutic proteins on a large-scale, commercial basis. However, the proteins expressed in tobacco usually need to be purified to high yield and purity from large amounts of biomass in order for their production to be commercially viable. The methods needed to purify proteins from tobacco are very challenging and not well studied. The objective of this research was to develop a process for the purification of the acidic model protein, recombinant β-glucuronidase (rGUS), from transgenic tobacco leaf tissue to high yield and purity. Polyelectrolyte precipitation with polyethyleneimine (PEI) was identified as an initial purification step for purifying acidic recombinant proteins from tobacco. Polyethyleneimine precipitation allowed for high recovery and concentration of the target protein while removing large amounts of impurities from the initial extract. At dosages of 700-800 mg PEI/g total protein, nearly 100% of the rGUS activity was precipitated with generally more than 90% recovered from the pellet. In addition, more than 60% of the native tobacco proteins were removed in the process, resulting in a purification factor near 4. Recombinant GUS was further purified by a step of hydrophobic interaction chromatography (HIC) followed by a step of hydroxyapatite chromatography (HAC). The HIC step served to remove PEI and other contaminants such as nucleic acids that were accumulated during the precipitation step, while the HAC step served to separate rGUS from the remaining native tobacco proteins, most notably ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). Nearly 40% of the initial rGUS activity was recovered as a near homogeneous fraction based on SDS-PAGE analysis after the three step process. The main steps used in this process are anticipated to be scalable and do not rely on affinity separations, making the process potentially applicable to a wide variety of acidic recombinant proteins expressed in tobacco as well as other leafy crops. / Master of Science