Chemical and Biomolecular Engineering - Theses
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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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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.