Chemical and Biomolecular Engineering - Theses
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ItemFunctional Supramolecular Network Engineering Inspired by Metal–Phenolic Complexation( 2021)Supramolecular assembly provides a versatile pathway for engineering bespoke materials, such as metal–organic hybrid materials. Metal–phenolic networks (MPNs), constructed from the coordination-driven assembly of phenolic ligands and metal ions, are an emerging class of hybrid materials with a rich choice of building blocks. Due to their strong adhesion to different substrates (particles, planar surfaces, microorganisms), high degree of modularity, and tuneable degradability, MPNs have garnered considerable attention in fields such as drug delivery, bioimaging, antimicrobials, separation, and catalysis. However, fundamental research in the material aspects of MPNs and how these influence biomedical applications are essential yet overlooked. This thesis explores the fundamental principles of MPNs and uses this insight to examine MPN materials in a range of biomedical applications. First, MPN microcapsules comprised of various building blocks are engineered, and their programmable gating mechanisms are explored in terms of intermolecular dynamics. This fundamental study not only provides insight into the dynamic nature of MPNs but also offers a route to engineer smart delivery systems and selective gating materials. Next, MPN coatings are used as a versatile and cytocompatible platform to trigger the endosomal escape of nanoparticles, which has been regarded as a key bottleneck for the intracellular delivery of therapeutics. The escape mechanism is systematically investigated and determined to be the “proton-sponge effect”, arising from the buffering capacity of MPNs. Notably, this buffering-enabled escape capability is preserved after the post-functionalization of MPN coatings with polymers, showing the generalizable nature of the platform. Therefore, a subsequent in-depth exploration of the buffering effects of MPNs sheds mechanistic insight into metal–organic systems and their emergent buffering capacity based on coordination dynamics and building block choice. Finally, the advantages of different polyphenol-enabled supramolecular networks are integrated to expand the MPN platform from thin films to self-assembled nanoparticles. Bioactive metal–phenolic nanoparticles are developed via robust and template-free assembly. Hydrophobic interactions and coordination play dominant roles in the assembly and stabilization of the nanoparticles. Furthermore, the incorporation of diverse biomacromolecules (e.g., functional proteins and genes) during assembly enables the potential use of these metal–phenolic nanoparticles in various biomedical applications, anticancer treatments, cascade reactions, and gene knockdown.
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ItemEngineering of DNA Micro- and Nanoparticles: Towards Vaccine Delivery( 2021)Vaccines are an effective tool for preventing and controlling various diseases by inducing adaptive immunity. Nanomaterials play an important role in vaccine development. Micro- and nanocarriers can be engineered to improve the therapeutic efficacy of vaccines by (i) preventing the degradation and systemic clearance of vaccine antigens and (ii) facilitating the uptake of vaccines in antigen-presenting cells (iii) co-delivering adjuvants and antigens at desired intracellular compartments for optimal immunotherapy. However, it is important to engineer a carrier that is both effective and safe. Micro- and nanoparticles based on DNA have shown great potential for biological applications, owing to the programmable sequences, predictable interactions, versatile modification sites, and high biocompatibility of DNA strands. This thesis aims to develop facile strategies to synthesize DNA particles for vaccine delivery by self-assembly approaches. First, a simple strategy to synthesize DNA microcapsules is reported. The cytosine-phosphate-guanosine oligodeoxynucleotides (CpG) motif is an efficient vaccine adjuvant that can effectively stimulate the immune system to secrete cytokines. By loading and crosslinking Y-shaped DNA building blocks (containing CpG motifs) into sacrificial calcium carbonate templates, monodisperse and spherical DNA capsules were obtained. These DNA microcapsules were internalized into cells efficiently, accumulated in endosomes, and induced immune cells to secrete high-level of cytokines. Next, we developed a template-assisted and versatile approach for synthesizing a new set of multifunctional particles through the supramolecular assembly of tannic acid (TA) and DNA molecules. Uniform and stable DNA-TA particles with different morphologies could be easily synthesized by using different types of DNA strands. Intriguingly, different DNA sequences can be encoded into this DNA-TA particle for applications in immunotherapy or gene delivery. The incorporation of CpG motifs and ovalbumin into the particles allows the intracellular antigen/adjuvant co-delivery to amplify cytokines production in macrophages, through synergistic effects. In addition, green fluorescent protein (GFP)-expressing plasmid DNA could be transfected by using the DNA-TA particles in HEK293T cells. Finally, nanometer-sized particles were engineered by exploiting the one-pot supramolecular assembly of TA, DNA, and PEG for intracellular delivery of CpG motifs. TA-DNA-PEG nanoparticles with different sizes could be fabricated by adding different molecular weight PEG chains. TA-DNA nanoparticles with tunable size were also synthesized by varying the molar ratio of TA and DNA. The obtained nanoparticles can enhance the cellular uptake of CpG oligonucleotides and consequently the production of cytokines in macrophages. Overall, the engineered DNA-based particles have potential for co-delivering nucleic acids and protein antigens in immune cells to enhance the immunological response against infectious diseases and cancer.
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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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ItemEngineering biomacromolecule-based particles with tunable functionality in biological systems( 2016)Particles with tailored physicochemical properties have numerous applications in diagnosis, therapy, and management of human diseases. In this context, elucidation of the interactions between biological systems and nanoengineered materials has emerged as an important research discipline, with the ultimate aim of controlling the interactions to achieve desired physiological responses. Biomacromolecules, such as peptides, proteins and polysaccharides, have diverse physiological functions, such as target recognition, signaling, and catalysis, which remain a challenge to mimic by synthetic methods. Biological systems precisely control synthesis, assembly, and disassembly of the biomacromolecules to guide physiological events. Therefore, controlled assembly of biomacromolecules into nano- and microscale particles may offer a promising platform to study bio-nano interactions, and ultimately to engineer functional materials for biomedical applications. However, previous studies have primarily been limited to particles assembled from biomacromolecules with little function. In this thesis, a robust strategy of assembling functional biomacromolecules into particles is developed, through the use of porous particles as sacrificial templates and reversible chemistry integrated into the biomacromolecular network. The advantages of this strategy include simplicity, versatility, tunability of particle morphology, triggered disassembly, and bioactivity that can be triggered in certain biological conditions. Three types of biomacromolecule-based particles were engineered: (1) peptide nanoparticles with proapoptotic activity (Chapter 3), (2) protein particles with pH-triggered recovery of enzymatic activity (Chapter 4), and (3) polysaccharide-based particles that can be targeted to tumour associated macrophages and Escherichia Coli (Chapter 5). These systems are used to demonstrate how the functionality of the particles in biological systems can be tuned using a chemistry and materials science-based approach.
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ItemIn vivo behaviour of polymer-based nanoengineered materials( 2016)Nanoengineered materials are attracting a great deal of interest as the basis for therapeutic delivery systems, due to their potential to prolong circulation half-lives, circumvent solubility problems and reduce toxicity through efficient targeting. The versatility of polymers and polymer-based materials makes them logical candidates in this area, where the ability to tailor particular functionalities is key to producing materials which have a place in the clinic. Specifically, capsules assembled using the layer-by-layer (LbL) technique offer unique control over material composition, size, shape and functionality. Additionally cylindrical polymer brushes (CPBs) offer unique properties, being single molecules which can offer particle-like dimensions through highly tuneable chemistry. Understanding the behaviour of such systems in vivo is critical to progressing materials beyond the laboratory. Achieving significant blood residence time is important for the ultimate bioavailability of potential encapsulated therapeutics. This thesis looks at the in vivo behaviour of both LbL assembled polymer capsules and cylindrical polymer brushes. Specifically this work aims to (i) investigate the behaviour of click-LbL capsule systems in vivo; (ii) extend the understanding of LbL capsule protein fouling behaviour, relating in vitro to in vivo findings; (iii) investigate the behaviour of cylindrical polymer brush materials in vivo. This will be demonstrated through the assembly of a range of click-LbL capsule systems including poly(methacrylic acid) (PMA), poly(N-vinyl pyrrolidone) (PVPON), and poly(2-diisopropylaminoethyl methacrylate) (PDPA), followed by tritium labelling and analysis using a rat model to establish capsule pharmacokinetics and biodistribution. The understanding of LbL capsule behaviour in vivo is then extended by applying poly(ethylene glycol) (PEG) functionalisation approaches to PVPON film and capsule modification. Capsules are functionalised using single PEG chains as well as densely grafted PEG CPBs. Methods used to assess film interaction with serum proteins in vitro are evaluated in light of in vivo performance. This leads to an in vivo study of CPBs to support their viability as drug delivery vehicles in their own right. CPB pharmacokinetics and biodistribution are shown to be dependent on both brush length and stiffness, with promising half-lives in the range reported for some stealth liposome systems. The fundamental in vivo data reported for both LbL capsules and CPBs are expected to form a valuable foundation for the further development of both systems.
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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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ItemAssembly of therapeutic carriers for sustained delivery of neurotrophins to the cochlea( 2013)Gradual degeneration of auditory neurons following sensorineural hearing loss is normally caused by a depleted supply of neurotrophins, as a result of the death of the cochlear hair cells, which secrete the signaling proteins. Animal studies show that the neurodegeneration could be prevented by exogenous administration of neurotrophins, which include brain derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), nerve growth factor (NGF) and neurotrophin -3 (NT-3). Optimum therapeutic benefit, however, requires continuous administration for a prolonged period. Although many novel delivery strategies (e.g. gene therapy, mini osmotic pumps and polymer hydrogels) have been previously proposed, none has been utilized at the clinical stage due to concerns about safety, cost and insufficient protein release. The primary focus of this thesis is the assembly and characterization of therapeutic carriers for the sustained delivery of neurotrophins to the cochlea. The study investigated two different carriers, namely capsosomes and calcium carbonate (CaCO3) supraparticles, by characterizing their loading capacities, as well as their long-term release kinetics in simulated physiological conditions, using lysozyme as a model protein and the BDNF. The capsosomes were prepared by incorporating liposomal compartments within hydrogel capsules using the layer by layer (LBL) assembly method, while the CaCO3 supraparticles were prepared by the evaporation initiated self-assembly of mesoporous calcium carbonates particles. The release of the proteins from the capsosomes was mainly dependent upon the composition of the liposomal compartments, while particles size and porosity governed the release from the supraparticles.
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ItemThe mechanical characterisation of nanostructured biomaterials( 2013)Hydrogel materials have demonstrated unique potential for biomedical application in areas ranging from macroscopic tissue engineering scaffolds to targeted nanoparticles for in vivo therapeutic delivery. Such propensity for biological application is largely due to the inherently high degree of hydration and low rigidity of hydrogel networks, which, being similar to those of natural tissue, often lead to good biocompatibility. The mechanics and nanostructure of such materials have been reported to have a significant effect on biological processes; from cellular interaction and fenestration clearance, to capillary flow and dynamics during circulation. This thesis examines novel methods forthe characterisation of both nanostructured planar and particulate hydrogel systems, using atomic force microscopy (AFM) force spectroscopy techniques in physiological buffer. The mechanical properties and material parameters for soft nanostructured biomaterials (thiol-modified poly(methacrylic acid) and poly(L-glutamic acid)) in various architectures (planar film, core-shell particle, free-standing capsule, and nanoporous particle) are herein investigated. It was found that a wide variety of soft structures could be characterised mechanically, and the corresponding results interpreted according to established theories and models for compressive deformation. As such, evaluation of the Young’s modulus for the hydrogel systems investigated in this thesis demonstrate the crucial role that system architecture and network density play in the resilience of soft structures to applied force. Compressive forces which occur in the biological domain, such as for cellular internalisation and soft-tissue cell retention, were subsequently linked to conclusions drawn from AFM measurements. Such preliminary investigations showed that both intrinsic and extrinsic properties of nanostructured hydrogels influenced cellular interaction; thereby forming the basis for further mechanobiological studies, allowing for the future rational design of nanostructured hydrogel biomaterial systems for in vivo biomedical applications.
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ItemAssembly of polymer matrices enveloping cubic lyotropic liquid crystalline nanoparticles for drug delivery applications( 2012)Cubic lyotropic liquid crystalline nanoparticles (cubosomes™) exhibit great potential as drug delivery vehicles due to their nanoscale size, biocompatible constituents, and high loading potential for hydrophobic, hydrophilic, and amphiphilic agents. However, they also suffer from some limitations which have restricted their clinical effectiveness. For example, they release their cargo in a rapid, uncontrolled manner— a phenomenon known as burst release. In addition, the lipids which form reverse cubic phase typically do not contain surface functional groups for the immobilisation of targeting or stealth providing moieties. Polymeric capsules, in particular those made with the layer-by-layer technique, are able to modify the release properties of a loaded drug according to the number and nature of polymer layers. Many of the polymers employed also contain available functional groups for additional chemistry. However polymeric capsules can be difficult to efficiently load with therapeutic agents, particularly when the drugs are lipid soluble. Additionally, the removal of the capsule core template often requires conditions that can cause instability. This thesis examined the use of polymers to modulate the properties of cubosomes with the intention to aid stability, limit burst release, add potential functionality, and increase the payload. Different methods used to prepare stable, well dispersed amphiphilic cubosomes (high pressure homogenisation, extrusion, and ultrasonication) were analysed and compared. The effect of an additive to the aqueous environment (such as sodium chloride or phosphate buffered saline (PBS)) was also investigated. Certain additives to the amphiphile matrix such as the charged lipids cetyl trimethylammonium bromide (CTAB), dioctadecyl-dimethylammonium bromide (DODAB) or sodium dodecyl sulphate (SDS) were found to cause structural changes to both bulk and dispersed cubic phase but could be tolerated up to a certain quantity before complete destabilisation occurred. Integrating cubic nanoparticles and polymer matrices was first accomplished by coating silica microparticles. This resulted in a multilayered polymer coating representing an embedded layer of cubosomes surrounded by poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) polyelectrolytes. Upon removal of the silica core, stable polymer microcapsules containing embedded cubic nanoparticles were obtained. A diversity of molecular encapsulation matrices is offered through the capsule core, polyelectrolyte layers, and the embedded cubosomes of these sub-compartmentalised, nanostructured microcapsules. Individual cubic nanoparticles surrounded by polyelectrolyte multilayers were prepared next. The polymers were able to interact with the non-charged cubic lipid nanoparticles by utilising a polyelectrolyte modified with hydrophobic side chains (poly(methacrylic acid-co-oleyl methacrylate), PMAO) as an initial layer. Three bi-layers of poly(L-lysine) (PLL) and poly(methacrylic acid) (PMA) were then sequentially added. In order to separate accrued polymer aggregates from the coated lipid nanoparticles, a simple technique was developed whereby centrifugation separated the less dense cubosomes for collection. Modulation of the drug release properties and attenuation of the burst release from coated cubosome particles was demonstrated using two model drugs (fluorescein and perylene). The modified polymer PMAO was then utilised as an alternative stabiliser for lyotropic liquid crystalline nanoparticles. The charge-stabilised particles were tested against the most commonly utilised steric stabiliser Pluronic F127 for stability and drug release characteristics. Although PMAO-stabilised nanoparticles still exhibited burst release, improved particle stability was observed over time and over a range of temperatures, including storage under refrigeration. A lesser amount of PMAO stabiliser and less energy input were also required to disperse the bulk lipid into discrete, uniform nanoparticles compared to Pluronic F127. These studies demonstrate the viability of combining layer-by-layer polymer matrix technology with cubic lyotropic liquid crystalline nanoparticles to enhance the future of drug delivery.
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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.