Chemical and Biomolecular Engineering - Theses

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    Nanoengineering Antibody Assemblies for Biomedical Applications
    Hu, Yingjie ( 2021)
    The clinical success of monoclonal antibody therapy has inspired research in understanding the fundamental molecular basis of antibody-antigen interactions and the engineering of antibodies with enhanced or novel properties. With the emergence of nanomedicine, antibodies have been widely applied as targeting ligands decorated on the surface of therapeutic nanostructured modalities – including liposomes, protein nanoparticles, and polymeric assemblies – for drug delivery and imaging applications. However, little is known about how antibodies assembled in a cluster or particulate form interact with antigens in a biological system, largely due to the challenge in preparing ‘pure’ antibody assemblies with controlled physicochemical properties. In this thesis, a mesoporous silica template-mediated assembly platform was applied to fabricate well-defined nano-assemblies of therapeutic antibodies, including conventional monoclonal antibodies and antibody-drug conjugates. The antibody nano-assemblies (AbNAs), crosslinked with poly(ethylene glycol)-N-hydroxysuccinimide (PEG-NHS), preserved the selectivity of the monoclonal antibody and induced receptor-mediated internalization of antibodies to achieve enhanced intracellular response, such as growth inhibition. This strategy presents opportunities for intracellular delivery of monoclonal antibodies, as well as a versatile platform for fundamental studies on the interactions between antibody assemblies and cells. Facile engineering of AbNAs can be achieved by leveraging the intrinsic property of the PEG crosslinker, such as chain architecture (PEG arm numbers and arm length), to regulate bio-nano interactions. As a widely recognized stealth material, PEG can prolong blood circulation time to allow the accumulation of nanoparticles in target tissues, however, it could also result in decreased targeting efficacy by blocking the antigen-binding sites. This thesis investigates the influence of PEG crosslinking, specifically the effect of using PEG crosslinkers with different chain architecture on the formation of AbNAs and their bio-interaction with respect to specific binding and uptake by phagocytic cells. PEG crosslinkers with less arms but longer arm length were found to be more beneficial for AbNAs to achieve both minimal phagocytic capture and optimal targeting. Furthermore, the targeting efficacy of AbNAs could be enhanced by substituting conventional monoclonal antibodies with engineered antibody fragments. Nanobodies, also known as single-domain antibodies (sdAb), are the smallest antigen-binding unit (12-15 kDa) that solely bind to the target antigen. The unique structure of nanobodies offers several desirable features, including small size, high stability, strong antigen-binding affinity and low immunogenicity, which makes nanobodies superior for antibody nano-assembly engineering. The nanobody nano-assemblies (NanoNAs), prepared via the template-mediated assembly platform, exhibited significantly enhanced selective association to target cells and reduced phagocytic association in comparison with full-sized AbNAs, owing to the unique structure of nanobodies that allowed a large amount of active binding sites to be presented on the particle surfaces and eliminated crystallisable fragment (Fc) receptor-mediated capture by phagocytic cells. Overall, the versatile antibody nano-assembly systems expanded our understanding of antibody-antigen interactions, and provides a facile platform to engineer antibody assemblies with novel or enhanced properties for biomedical applications.
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    Engineering Particle Systems for Pulmonary Delivery
    Song, Jiaying ( 2020)
    Pulmonary delivery has proven to be a promising delivery route for either local lung targeting or systemic delivery. A variety of particle systems such as polymeric particles and lipid-based particle systems have been developed as therapeutic delivery carriers for pulmonary delivery. However, the majority of current inhaled particles have limited retention time and low bioavailability in the target lung region, leading to suboptimal efficacy of therapeutic delivery and needing increased drug dosage or dose frequency, which could cause severe side effects. This is mainly due to the clearance and metabolic degradation mechanisms in the lungs. The mucociliary clearance and the alveolar macrophage clearance defend the airway and deep lung region, respectively, and are responsible for the elimination of inhaled particles. Therefore, it is important to understand the interactions of particles with the complex lung physiological environment in order to design more effective drug delivery carriers that can overcome various biological barriers or exploit the defence mechanisms to achieve improved biological outcomes. This PhD thesis focuses on engineering particle systems for pulmonary drug delivery, with specific aims of studying the interactions between inhalable particles and complex biological systems in the lungs including particle–mucus interactions and the role of a pulmonary corona in the uptake or clearance of particles by alveolar macrophages. Poly (ethylene glycol) (PEG) as a low-fouling material commonly used for ‘stealth’ modification of particles to reduce immune clearance was first investigated. The use of PEG building blocks with various architectures resulted in PEG-based particles with different structures and mechanical properties, which further affected the interactions of particles with proteins and immune cells in a complex biological environment (e.g., human blood). The particle–mucus interactions were then studied in the second part of this PhD research by comparing different polymer particles with potentially mucoadhesive and mucus-penetrating properties, obtaining a basic understanding of mucociliary clearance of particles in the lungs. The role of the pulmonary protein corona in alveolar macrophage clearance of polymer particles was then studied. The presence of a protein corona on particles resulted in increased or reduced macrophage uptake depending on the particle properties. When particles were transferred from one biological environment into another (e.g., blood to lungs), the interplay of protein coronas formed in each environment determined the composition of the eventual mixed protein corona and the subsequent particle–cell interactions. Finally, drug (structurally nanoengineered antimicrobial peptide polymers) loading and intracellular delivery using promising polyphenol-based carriers were investigated as potential antimicrobial therapies against lung infections (e.g., tuberculosis).