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.