Chemical and Biomolecular Engineering - Theses
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ItemMaterial-based gene therapy approaches for HIV and neurodegenerative diseases( 2020)Gene therapy is of interest in medicine as it allows potential treatment of inherited and acquired diseases that cannot be treated or prevented using conventional methods. The introduction of new genetic material into the cells aims to improve cellular functions by either replacing a malfunctioning gene with a functional transgene or silencing the expression of specific genes implicated in various human diseases. The delivery of plasmid DNA provides an opportunity to replace defective or missing genes by utilizing cellular gene expression apparatus to produce encoded proteins. RNA therapeutics act via the RNA interference pathway to target intermediate gene expression product for degradation and prevent its translation to protein. Free nucleic acids typically experience rapid blood clearance and a short circulation lifetime and are unable to cross biological membranes due to electrostatic repulsion between DNA/RNA phosphate groups and phospholipids in the cell membrane. Therefore, there is a need to formulate gene carriers for improved pharmacokinetics of DNA/RNA therapeutics and efficient delivery to the site of action. The main objective of this research project was to develop material-based systems for gene delivery and apply it to HIV therapy and Friedreich’s ataxia (FRDA). Polyarginine-containing capsules were prepared via layer-by-layer assembly and enabled efficient complexation of anti-HIV siRNA. The functional effect via transcriptional gene silencing of the viral genome was demonstrated in virus-infected primary cells. To investigate how cellular changes associated with cell activation and viral infection influence the particle-cell interactions, particles association with activated primary cells and pseudovirus-infected T cells was investigated. In the second part of the thesis, the optimization of DNA binding by polyarginine-containing LbL core-shell particles and the delivery of frataxin-encoding plasmid DNA to address the FRDA-associated frataxin depletion was demonstrated using patient-derived iPSC neurons. The role of particle size, charge and density in the interaction of particles with iPSC 3D neuronal organoids was also demonstrated. This thesis presents the preparation and characterization of LbL-assembled particles as a versatile system with easily tailorable properties and its application in gene therapy for viral and neurodegenerative diseases. The presented research also aims to gain a fundamental understanding of bio-nano interactions in various biological systems.
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ItemThe development of thin films for efficient carbon capture and storage( 2016)The chemical absorption of carbon dioxide (CO2) into a monoethanolamine (MEA) solvent is the accepted commercial process for industrial CO2 capture operations. Gas-liquid absorption processes are conducted mainly in absorption towers or membrane contactors, with absorption columns being the preferred industrial method of choice. However, the subsequent desorption of CO2 from the typical MEA solvent is extremely energy intensive. Alternative solvents, such as potassium carbonate (K2CO3), are more energy efficient and relatively inert in comparison to the alkanolamines, but their slow reaction kinetics in the absorption process limits their application in absorption processes. A membrane contactor is preferable to a packed column for absorption because of its large surface-to-volume ratio and its ease in scaling up. The membrane gas absorption (MGA) of CO2 makes use of a porous hydrophobic membrane material as a barrier between a CO2 gas stream and an aqueous solvent stream to absorb the gaseous CO2 into the liquid solvent. The CO2 diffuses across the membrane and dissolves into the solvent, where it can subsequently be stripped from the solvent for solvent reuse and storage of the CO2. However, most membrane materials are easily wetted by the alkanolamine solvents, resulting in reduced mass transfer rates. Hence, membrane contactor operations are more preferably run with solvents that are not as likely to cause mass transfer issues associated with wetting as the alkanolamines do. The use of a carbonic anhydrase (CA) enzyme as a reaction promoter may overcome the reduced overall absorption rates exhibited by K2CO3 relative to MEA. However, these enzymes tend to denature at higher temperatures and would not be suited for use in circulation within a traditional absorber-stripper process. The immobilization of the enzyme within the gas absorber or onto a membrane contactor can increase enzyme stability and avoid thermal denaturation in the stripper. However, immobilization is only effective if the mass transfer of CO2 through the liquid phase to reach the immobilization substrate does not become rate controlling. The enzyme-aided mass transfer of CO2 is at its most effective when the enzyme is immobilized at the gas-liquid interface, and the use of porous microparticles containing adsorbed CA in an absorption column has not yielded much results either. The immobilization of CA onto a membrane contactor brings it closer to the gas-liquid interface and has yielded more substantial increases in the mass transfer kinetics. Multiple methods for immobilizing CA onto the surface of a membrane contactor have been proposed, though the layer-by-layer (LbL) technique for enzyme immobilization has not been investigated for enhancing CO2 absorption operations thus far. LbL assembly has been used effectively on a wide range of polyelectrolytes and proteins for the fabrication of uniform thin films on different planar surfaces. It is possible to immobilize CA in thin films on a polymeric membrane surface for enhancing CO2 mass transfer rates to ensure that the CA is at the gas-liquid interface. Enzyme immobilization stabilizes the enzyme structure and preserves its activity over a long period of time, and multiple layers of enzyme can be added to increase the enzyme loading on the membrane surface. However, the coating of polyelectrolytes onto a surface renders the surface more hydrophilic and closes up the pores of the membrane surface, which may cause wetting to occur more easily or the mass transfer to be more hindered by the polyelectrolyte layers. It was found in this investigation, though, that the deposition of the CA as a thin film on the surface of a flat sheet membrane was able to increase the membrane resistance to wetting by closing up the membrane pores significantly, even though the hydrophilicity of the membrane was increased after the assembly of an ultrathin film on the membrane surface via LbL adsorption. The layering of mesoporous silica nanoparticles onto the membrane surface further helped to close up the pores and increase the total enzyme loading onto the flat sheet membrane as compared to a regular multilayer enzyme film. However, the specific enzyme activity was found to be significantly reduced upon the immobilization to the nanoparticles. Hence, it was decided that the LbL film assembly technique would only make use of regular multilayer films when scaling up immobilization onto hollow fiber membranes. The scaling up of the LbL technique to hollow fiber membranes also yielded similar results after fabricating a multilayer film on the surface of the membrane, where the CO2 mass transfer rates were significantly increased after coating the hollow fibers with thin polyelectrolyte film layers. An increase in the number of CA layers coated onto the membrane corresponded with an increased mass transfer coefficient in the transport of CO2 across the hollow fiber membranes, as did a reduced flow rate or an increased polyelectrolyte adsorption contact time. Both porous polypropylene (PP) and nonporous polydimethoxysilane (PDMS) membranes were tested in contactor operations. The degree of pore wetting on the PP was also investigated to determine its effects on the overall rates of mass transfer. Any feasibility study regarding the use of the enzymes always has a concern regarding the operational lifespan of the enzyme. Nonporous PDMS hollow fiber membranes were used to separate the effects of pore wetting from enzyme deactivation in monitoring the decline of the mass transfer coefficient over time. The hollow fibers were operated at elevated temperatures of 35°C and 50°C to determine the survivability of the CA in the membrane contactor at those temperatures. The CA was found to be completely deactivated after 80 days of exposure to 30 wt% K2CO3 at 25°C, while its deactivation was complete after only 3 days of exposure to 30 wt% K2CO3 at 50°C. The immobilized CA was also contacted with toxic gases such as nitric oxide (NO) and sulfur dioxide (SO2) or their associated nitrate (NO3-) and sulfate (SO42-) ions in solution, as these are components of post-combustion gas streams that can inhibit the activity of the CA. The exposure of the immobilized CA to the dry gases or their associated anions did not significantly affect the activity of the immobilized CA. It can therefore be said that CA is a useful promoter for the absorption of CO2 over a short term period. However, its stability under harsh operating conditions has to be improved for further long-term operational feasibility.
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ItemRapid, facile and automated polymer assembly techniques for the preparation of layer-by-layer capsules( 2014)Layer-by-layer (LbL) assembled films and capsules have shown potential application in diverse fields such as energy and drug delivery, because they can be prepared from various polymers and loaded with numerous materials. Although film deposition on planar substrates can be rapid and facile using techniques such as spray-coating or spin-coating, the layering of particles generally requires longer and more involved protocols. LbL assembly on particles typically uses random diffusion as the adsorption driving force, which can lead to the risk of aggregation during layering and washing. To overcome the inherent challenges surrounding polymer deposition on particulate matter, immobilization techniques for suspending the template particles were developed. Immobilizing the template particles has allowed for rapid, facile, and automated layering methods to be applied to particles of varying sizes and compositions. Large particles were suspended in a liquid-based fluidized bed for layering, while smaller particles required immobilization in a porous hydrogel before facile layering was possible. The use of a fluidized bed allowed for the large scale production of polymer microcapsules at a rate roughly ten times faster than conventional methods. However, this technique was generally limited to particles above 5 micrometers. For the hydrogel immobilized particles, a naturally derived polysaccharide, agarose, was used as an immobilizing agent. Electrophoresis, convection and diffusion were then used to deposit polymers on immobilized particles and form capsules ranging from below 100 nanometers to above 1 micrometer. These driving forces allow for the use of different polymer combinations, can be used to load various types of cargo, and can be used to form polymer replica particles. Each driving force has unique benefits, and all three driving forces are either already automated, or potentially automatable. The speed, ease, scope and scale with which these capsules can now be produced should benefit research and development directed towards the application of LbL capsules.
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ItemNanoengineered switchable, multi-responsive carriers for biomedical applications( 2014)Recent progress in material science and nanotechnology has enabled the design of next-generation drug delivery carriers. The implementation of such delivery systems has the potential to significantly enhance the current treatment outcomes, owing to their ability to achieve targeted bio-distribution and enhanced drug payloads. To develop the next generation of drug carriers, it is critical to incorporate a stimuli-responsive trigger into the material design. This allows for the development of “smart” carriers, which can load and release therapeutics in a specific targeted site on demand. Poly(2-diisopropylaminoethyl methacrylate) (PDPA) is a stimuli-responsive polymer that undergoes reversible hydrophobic-hydrophilic phase transition at biological-relevant pH variations. The incorporation of PDPA in the drug delivery systems opens a new route toward advanced drug delivery applications. This thesis focuses on developing several bottom-up approaches to assemble PDPA-based stimuli-responsive delivery systems from a material science perspective. By utilizing Layer-by-Layer (LbL) and self-assembly techniques, switchable, multifunctional systems that responded to various cellular conditions were synthesized. Charge-shifting PDPA capsules were synthesized via LbL assembly and cross-linked using a redox-responsive cross-linker. Dual stimuli-responsive cargo release profiles by pH and redox change were assessed in simulated intracellular conditions. Intracellular degradation kinetics of these capsules was investigated. The tuning of degradation kinetics was achieved by varying the degree of cross-linking density in the capsules, as confirmed by radio scintillation counting. Novel cross-linker free PDPA capsules were later developed. It was found that these capsules could improve the loading ability of drugs as small as 500 Da, and rapidly deconstruct and release cargo upon cellular uptake. Moreover, utilizing self-assembly techniques, multifunctional nanoparticles were synthesized from blending PDPA with an anti-cancer drug and a cell penetrating peptide. By varying the loading ratio in the nanoparticles, tunable cytotoxicity up to 30-fold was achieved. The reported PDPA-based responsive carriers are expected to provide fundamental insights towards the rational deign and synthesis of advanced delivery systems.
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ItemTunable polymer capsules for therapeutic delivery applications( 2013)The area of therapeutic drug and gene delivery has made rapid progress over the last two decades. This interest in the development of drug delivery systems is aimed at protecting the body from the non-specific side effects of drugs and biomolecules; protecting therapeutic compounds from premature degradation and directing them specifically to target sites; and having tunable carrier degradation and cargo release to optimise the therapeutic efficacy. Of the drug delivery systems studied, the Layer-by-Layer (LbL) approach for the assembly of polymer multilayer films and capsules is of particular interest because it is a facile and highly versatile assembly technique that allows for specific properties to be tailored into the assembled materials to fulfill various criteria needed for the successful bioapplication of these systems. This thesis focuses on the development of a biologically compatible polymer capsule system based on the LbL technique and the incorporation of chemical and physical approaches to achieve stable cross-linked capsules, cargo loading and controlled release. Specifically, the work aims to (i) study the film build up and characteristics of cross-linked polymer films and capsules; (ii) develop stable polymer capsules capable of loading a cargo; (iii) demonstrate a modular approach in achieving tunable capsule degradation and cargo release; and (iv) provide some insight into the behaviour of these capsules in in vitro cell studies. This is demonstrated through the fundamental studies of the assembly of a low-fouling poly(N-vinyl pyrrolidone) (PVPON) capsule which uses a cross-linker in film stabilisation. The cross-linker has a reducible disulphide bond that endows the capsules with stimuli-responsive degradable properties. The optimised capsule system is loaded with a model plasmid DNA and used to demonstrate tunable carrier degradation and cargo release behaviour, through the use of the cross-linkers, in simulated cellular reducing environment. The study is extended to the assembly of hybrid systems, which is based on incorporating a charge-shifting polymer poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) and a block copolymer poly(2-(methacryloyloxy)ethyl-phosphorylcholine)-b-poly(2-(diisopropyl-amino)ethyl methacrylate) (PMPC-PDPA), into films and capsules. These hybrid systems are investigated for their properties. Finally, these different capsule systems are investigated for their interaction and internalisation behaviour in in vitro cell studies. The preliminary results offer some insight into the potential use of these highly engineered drug delivery carriers in biomedical applications.
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ItemPoly(methacrylic acid) hydrogel capsules as a platform for biomedical applications( 2012)The design and assembly of biocompatible nanoengineered carriers is of interest due to their potential applications in biotechnology as tools for catalysis and sensing, in biomedicine as systems for drug delivery, in diagnostics, and in vivo imaging. The layer-by-layer (LbL) technology is a prominent technique to design carrier systems for biomedical applications. In recent years, poly(methacrylic acid) hydrogel capsules (PMA HCs), based on disulfide-stabilized poly(methacrylic acid), have been fabricated from the LbL technique, and thoroughly studied to gain control over their stability, degradability and cargo release. These capsules are obtained by the sequential deposition of thiolated poly(methacrylic acid) (PMASH) and poly(N-vinylpyrrolidone) (PVPON) onto silica particles via hydrogen bonding. Upon controlled crosslinking and removal of the silica template, PVPON is released at physiological conditions due to the disrupted hydrogen bonding between PMASH and PVPON, which results in single-component PMA hydrogel capsules. This work provides an insight into designing a novel architecture and biofunctionalization of PMA HCs for enhanced and targeted drug delivery. Specifically, this research describes novel hydrogel capsule architectures, namely subcompartmentalized hydrogel capsules (SHCs), which are designed for potential applications in drug delivery and microencapsulated biocatalysis. Examples of SHCs with tens of subcompartments are demonstrated with their successful drug/cargo loading, as well as selective degradation of the SHC carrier and/or sub-units in response to multiple chemical stimuli. To develop a facile surface functionalization approach of the PMA hydrogel capsules, retention of PVPON was employed through modification of the polymer to obtain a bifunctional polymeric linker. The antibody-functionalized PMA/PVPON HCs demonstrate significantly enhanced cellular binding and internalization to specific cells, suggesting these capsules can specifically interact with cells through antibody/antigen recognition. To understand the impact of aspect ratio on cellular function, PMA HCs were prepared with various aspect ratios. Careful control over aspect ratio of the silica rods provided the ability to control the aspect ratio of the PMA HCs. Upon incubation of these capsules with living cells, varied behavior was observed, suggesting different mechanisms for their interactions with cells.
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ItemCapsosomes: en route toward synthetic cellular systems( 2011)Engineering artificial cells is currently an emerging area of research that involves constructing mimics of biological cells. These biomimetic cellular structures hold tremendous promise for the creation of next-generation therapeutic tools due to their ability to restore lost cellular functions. Amongst their potential applications, replenishing absent or malfunctioning enzymatic activities to degrade waste products or to support the synthesis of medically relevant biomolecules is a chief goal, which can provide long-term therapeutic solutions for chronic diseases. Artificial cells do not require the complex multifunctionality of their biological counterparts and can be more simply designed to perform a specific activity. A key approach in designing a cell-like system is a subcompartmentalized assembly, which is one of the features of biological cells that enable the performance of multiple complex biochemical reactions within confined environments. This thesis focuses on developing a bottom-up approach to assemble micron-sized vessels with a controlled number of enzyme-loaded subcompartments toward cell mimicry. Capsosomes, polymer hydrogel capsules containing controlled amounts of intact cargo-loaded liposomal subcompartments, were developed in this thesis and they represent a novel class of carrier system toward the design of bioinspired vehicles. Polymer capsules, assembled via the sequential deposition of interacting polymers onto particle templates (layer-by-layer technique, LbL) followed by core removal, serve as structurally stable scaffolds with tunable permeability that allow exchange of reagents and nutrients between the internal and external milieu – resembling cell membranes. On the other hand, liposomes divide the interior of the capsules into subcompartments and can stably encapsulate fragile hydrophobic and hydrophilic cargo, e.g., enzymes in order to conduct encapsulated catalysis – resembling cell organelles. The creation of (bio)degradable capsosomes is based on the sequential assembly of cargo-loaded liposomes and polymers onto sacrificial particle templates, followed by the LbL deposition of poly(N-vinylpyrrolidone) (PVP) and thiol-functionalized poly(methacrylic acid) (PMASH) via hydrogen bonding. Upon crosslinking the thiols of the PMASH and dissolution of the particle templates, colloidally stable capsosomes are obtained. The coassembly of polymers and liposomes was optimized via novel noncovalent linkage concept using tailor-made cholesterol-modified polymers and this unique approach facilitates stable incorporation of intact liposomes into polymer films. Spatial position of the subcompartments can be controlled, which yields capsosomes containing membrane-associated or “free-floating” subunits. Capsosomes exhibit size-dependent retention of the encapsulated cargo within the liposomal subunits. To prolong the stability of the liposomes in the compartmentalized assembly against degradative enzymes, the outer membrane of the capsosomes was surface functionalized with poly (ethylene glycol) (PEG). The functionality of capsosomes was demonstrated by triggered encapsulated (two-step) enzymatic catalysis. Capsosomes encapsulating glutathione reductase were able to generate glutathione, a potent antioxidant, while simultaneously releasing small therapeutic molecules, which highlights the ability of this subcompartmentalized assembly in addressing the complexity in therapeutic cell mimicry. The phase transition temperature of the liposomes was used as a trigger to initiate the enzymatic reactions, allowing capsosomes to be repeatedly used for multiple subsequent catalysis. Capsosomes with tailored properties present new opportunities en route to the development of functional cell mimics and the presented studies highlight crucial aspects for the successful applications of capsosomes as therapeutic artificial cells.
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ItemBiologically compatible thin films by click chemistry( 2010)The assembly of materials by the Layer-by-Layer (LbL) method provides a versatile and flexible method of assembling nanoscale structures with tailored composition, thickness, size, shape and functionality. Many conventional methods of assembling materials such as thin films and hollow polymer capsules fall short in achieving versatile, covalently bound multilayers. Combining click chemistry, a highly efficient and specific set of reactions, with the established LbL technique provides a number of advantages for the preparation of thin films and hollow polymer capsules. The click-LbL approach permits the production of stable, tailored and functionalisable materials through the modular assembly of a variety of building blocks. This thesis focuses on developing biologically compatible thin films and capsules by the click- LbL technique. Specifically, this work aims to: (i) investigate the fundamental factors controlling the growth and characteristics of click-LbL films; (ii) develop stable, biocompatible films; (iii) demonstrate that click chemistry is a facile and flexible means of functionalising multilayer films; and (iv) demonstrate that click-LbL films are capable of performing specific roles within biological systems. This will be demonstrated through a fundamental study on the assembly of thin films and capsules through assembly of alkyne- and azide-functional poly(acrylic acid) building blocks by the click-LbL technique. The technique is then extended to the assembly of covalently bonded films comprised solely of poly(ethylene glycol) (PEG) acrylates that limit the adsorption of serum proteins. By functionalising click-LbL films of PEG with a cell adhesion promoting peptide, these films then demonstrate the specific adhesion and growth of cells onto a surface. Multilayer films are also assembled using temperature-responsive PEG methacrylate copolymers and their ability to resist the adsorption of proteins from human serum is tested. Hollow capsules comprised of poly(N-vinyl pyrrolidone), with demonstrated low-cytotoxicity, are then produced by multilayer assembly onto colloidal silica. These capsules are covalently stabilised through a click-functionalised cross-linking agent, which can be cleaved under reducing conditions, to deconstruct capsules. The demonstrated properties of click-LbL films and capsules presented herein, offer potential for chemical engineering, pharmaceutical and in particular, biological applications.