Chemical and Biomolecular Engineering - Theses
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ItemApplication of Advanced Fluorescence Imaging Techniques for Intracellular Tracking of Nano-biomaterials( 2021)The engineering and intracellular delivery of nanoparticles with tailored structural, functional and therapeutic properties is challenging due to the interactions of nanomaterials with complex and dynamic biological systems. Additionally, the clinical translation of nanoparticle-based chemotherapeutics is hampered by the poor capacity of 2D cell monolayer culture to mimic in vivo tumour microenvironment and cell-cell interactions. To overcome these biological barriers and enable the clinical translation of nanoparticles, a thorough investigation of nanoparticle-cell interactions in complex biological environments is of paramount importance. For this purpose, novel advanced fluorescence techniques enable the study of nanoparticles structure and functional properties inside the cells with improved spatial and temporal resolution. Additionally, the development of complex 3D cell culture systems mimicking tumour tissue could provide a novel method to predict the in vivo behaviour of nanoparticle-based chemotherapeutics and cellular response to the treatment. Herein DNA- and sugar-based nanoparticles have been developed as platforms for the detection of molecular targets and delivery of drugs within cells and in complex biological settings. Specifically, fluorescence resonance energy transfer microscopy, fluorescence correlation spectroscopy, fluorescence lifetime imaging microscopy and multicolour single-molecule localization microscopy were employed to probe the specific binding of the DNA nanosensor to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and the nanocomplexation of glycogen with albumin. The intracellular trafficking and the activity of drug-loaded nanoparticles were investigated in 2D and 3D static and dynamic cell culture systems. The biological activity of glycogen-albumin nanoparticles was investigated in a 3D tumour microtissue obtained by co-culturing BT474, NIH-3T3 and RAW264.7 cells in a U-Cup perfusion bioreactor device. The interactions of glycogen-albumin nanoparticles with peripheral blood mononuclear cells isolated from human blood and nanoparticles in vivo biodistribution in mice were also analyzed. This study aims to gain an understanding of the bio-nano interactions in various biological systems and highlights the importance of combining multiple fluorescence techniques and complex models for monitoring the intracellular behaviour of nanomaterials and accurately predicting their in vivo behaviour.
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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.