Chemical and Biomolecular Engineering - Theses
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ItemEnzyme-responsive nanomaterials for the delivery of antimicrobial peptides( 2023-10)The rate of resistance to antibiotics that are commonly used in the clinic is escalating rapidly, surpassing the introduction of new antimicrobial drugs. To address this problem, alternative strategies are being explored, such as the re-evaluation of antibiotics, that have not yet gained widespread clinical application. Antimicrobial peptides (AMPs) represent one of those antibiotics, offering remarkable antimicrobial efficacy against various pathogens. However, in clinical settings, AMPs are typically considered a last-resort option due to their off-target effects and poor stability in-vivo resulting from their cationic and amphiphilic peptide nature. Therefore, most of current strategies addressing these limitations focus primarily on the control and shielding of the cationic charge and the amphiphilic nature of AMPs. These can potentially be achieved through encapsulation of AMPs inside stimuli-responsive polyelectrolyte complexes (PECs) by combining the cationic drug with anionic polyelectrolytes. Stimuli-responsive polymers can be employed as encapsulation materials in PECs to design systems that activate drug release in response to specific changes encountered during microbial infection, such as variations in pH, enzyme activity, or temperature. The overarching aim of this Thesis was to explore the creation of PECs capable of encapsulating the clinically approved antimicrobial peptide, Polymyxin B and its subsequent enzyme-induced release. In Chapters 2 and 3, the aims were: Firstly, to synthesize anionic and helical polymers incorporating enzyme-degradable peptide side chains (Aim 1.1), followed by evaluation of the degradation properties of these polymers in response to the enzyme released by gram-negative bacterium Pseudomonas aeruginosa (Aim 1.2). In Chapter 4, the first objective (Aim 1.3) was to assemble Polymyxin B and the anionic enzyme-degradable polymers into PECs. The next objective (Aim 1.4 ) involved investigating the P. aeruginosa-induced drug release from these PECs, while Aim 1.5 focused on assessing the antimicrobial activity of the developed PECs against P. aeruginosa strains. Chapter 1 provides a review of the current developments in the field of the stimuli- responsive delivery of AMPs using polyelectrolyte complexes. Chapters 2 and 3 discuss the synthesis of polymers with poly(methacrylamide) and poly(acetylene) backbones respectively coupled to enzyme-degradable peptide side chains. Of the synthesized materials, two poly(methacrylamide) polymers from Chapter 2 were found to be the most effective in terms of degradation by the enzyme released by P.aeruginosa, while none of the synthesized acetylene-containing peptides from Chapter 3 polymerized. Subsequently, the poly(methacrylamide) polymer with the highest multivalency was used to form PECs with Polymyxin B in Chapter 4. Eight different formulations of PECs were created, with one being the most optimized in terms of encapsulation efficacy and physiological stability. The stability of the particles was further improved by the addition of Tannic Acid, which acted as a protective coating and a cross-linker. The Thesis then evaluated the ability of the PECs to release Polymyxin B under enzymatic degradation. Finally, the preliminary evaluation of the antimicrobial activity of the PECs against various P.aeruginosa strains were presented.
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ItemSynthesis of DNA-inspired polymers with nucleotide-pendant functionalities( 2023-04)Deoxyribonucleic acid (DNA) is the most important hereditary material. This natural polymer possesses an outstanding ability to store and translate genetic information. However, its use in the biosciences has been restricted by the high cost involved in producing long DNA sequences with more than 200 base pairs, and the limited shelf life of these sequences once produced. To tackle the stability and cost related issues, developing DNA-like materials, such as DNA-inspired polymers, is of increasing interest. Furthermore, combining the DNA substituents and synthetic polymers provides special features that are not seen in either material alone. For example, synthetic polymers typically do not display defined structures and functions as DNA. One of the major challenges in generating DNA-inspired materials is the inclusion of the entire DNA building block into the system, namely the nucleotide, consisting of the nucleobase, deoxyribose and phosphate groups. This project aims to combine all three DNA components into a polymeric system. Exploring the synthetic routes and studying the specific interactions and behaviour of the produced system are the major aims of the project. To accomplish these aims, first adenine (A), thymine (T), cytosine (C) and guanine (G) protected nucleotide functionalised propyl methacrylate monomers were synthesised. This was achieved by coupling 2-hydroxy propyl methacrylate (HPMA) to commercially available protected phosphoramidites, 5'-dimethoxytrityl-N-benzoyl-2'- deoxyadenosine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (hereafter denoted as dA(Bz)), 5'-dimethoxytrityl-N-benzoyl-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (hereafter denoted as dC(Bz)), 5'-dimethoxytrityl-2'-deoxythymidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (hereafter denoted as dT) and 5'-dimethoxytrityl-N-isobutyryl-2'-deoxyguanosine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (hereafter denoted as dG(ib) via phosphite triester linkages. By designing bio-functional monomers, containing protecting groups, side-reactions were minimised and increased solubility in a broad range of solvents was realised for subsequent polymerisation reactions, i.e., free radical polymerisation (FRP) and reversible addition fragmentation (RAFT). This is the first time phosphoramidite-HPMA monomers have been synthesised via a room temperature approach, and isolated for polymerisation reactions. Secondly, the seminal synthesis of A and T phosphoramidite-functionalised propyl methacrylate polymers was realised, using post- and pre-functionalisation methods via RAFT polymerisation. The RAFT method was used to achieve a higher control over the molecular weight of the produced polymers. Using the post-polymerisation functionalisation method (addition of phosphoramidite functionalities to already polymerised HPMA polymer), a RAFT-made poly(HPMA) with a narrow dispersity (1.29) and controlled DP (58), was functionalised with dA(Bz) and dT protected phosphoramidites via phosphite triester linkages. Although the polymer synthesis was successfully performed, the degree of phosphoramidite functionalisation was low, particularly for the A containing polymer (45 %). Pre-polymerisation functionalisation (direct polymerisation of the HPMA nucleotide-based monomers) was then used to investigate if there was any improvement in the degree of phosphoramidite functionalisation along the polymeric backbone. Applying this approach, A and T phosphoramidite-HPMA monomers were directly polymerised using the RAFT technique. The attempts to improve the degree of functionalisation was successful in case of A containing polymer (66%), however, the degree of functionalisation remained approximately the same as the T containing polymers for both methods (57% for post- and 50% for pre-functionalisation processes). These polymers offer some unique features which are currently not seen in literature including relatively high degree of protected nucleotide functionality, ready dispersion in solvents appropriate to a broad range of polymerisation and protection of the amine group on the nucleobases, ideal for RAFT polymerisations. In addition, the RAFT polymers, still with pendant RAFT agent, have future potential as macro-RAFT agents for subsequent block extension through the addition of other monomers for potential sequence control. This is not explored within the scope of this thesis. The final part of the thesis the noncovalent interactions of deprotected A and T nucleotide-functionalised propyl methacrylate polymers in solution was studied. To achieve this, the removal of the phosphoramidite protecting groups was required to achieve free hydroxyl and amino functionalities which could further interact via noncovalent bonding. The resulting polymers were potentially capable of noncovalent interactions with their complementary polymer (A with T). The noncovalent interactions between the A and T deprotected nucleotide-functionalised propyl methacrylate polymers were studied using ultraviolet (UV) and circular dichroism (CD) spectroscopies at various temperatures. These studies revealed that the pi-pi stacking between the complementary A and T nucleobases was the dominant interaction in the polymer mixture. Performing the studies in three different solvents (dichloromethane (DCM), cyclohexane (CY) and isopropanol (IPA)) showed that increasing the solvent dielectric constant promotes the pi-stacking interactions. For the A and T deprotected nucleotide-functionalised propyl methacrylate polymer mixture in IPA, the enhanced pi-stacking led to spherical and worm-like polymeric assembled structures with average diameters of 100 nm (confirmed by transmission electron microscopy (TEM)). The morphology of the particles was controlled by the degree of functionalisation, where the post-functionalised polymers formed worm-like particles and the pre-functionalised polymers formed spherical particles. In summary, this thesis showed the RAFT synthesis of nucleotide functionalised propyl methacrylate polymers, including complementary nucleobases A and T. The inclusion of hydrophobic and hydrophilic groups along the polymeric backbone led to the formation of self-assembled polymeric nanostructures. The specific structure and behaviour of the produced DNA-like materials makes them attractive for a broad range of biomedical applications such as delivery of drugs and genetic materials. Moreover, the inclusion of phosphate linkages into these materials paves the way for the synthesis of polymers with improved helicity levels.
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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.