Chemical and Biomolecular Engineering - Theses
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ItemEngineering glycogen-siRNA constructs with bioactive properties( 2019)RNA therapeutics, such as small interfering RNA (siRNA), have great potential for the treatment of inherited and acquired diseases that are not curable with conventional methods. The delivery of new genetic material into cells provides an opportunity to alter the expression of malfunctioning genes. However, siRNA is a hydrophilic and negatively charged molecule, which cannot easily cross biological membranes and is susceptible to degradation by nucleases present in biological fluids. Therefore, siRNA therapeutics require carriers that can effectively deliver their cargo into target cells. Early formulations for siRNA delivery involved systems based on viral vectors, lipid-based nanoparticles and cationic polymers. However, these formulations often displayed high toxicity, immunogenicity, instability in biological media, inability to penetrate tissue, and/or rapid clearance from the blood stream. Fine control over carrier size and surface properties, use of simplified and reproducible synthesis approaches, and deeper understanding of the interactions between siRNA-nanoconstructs in extra- and intracellular environment can potentially improve the engineering of new carriers. In this thesis, influence of the structural properties of soft glycogen nanoparticles on the formation of siRNA constructs and their delivery in a complex biological environment were investigated. Glycogen is a hyper-branched glucose bio-polymer of nanometer size that may be isolated from various animal tissues or plants. It is composed of repeating units of glucose connected by linear α-D-(1−4) glycosidic linkages with α-D-(1-6) branching. In this work, the properties of soft glycogen nanoparticles were tailored for the engineering of glycogen-siRNA constructs. These constructs were carefully designed to efficiently penetrate 3D multicellular tumour spheroids and exert a significant gene silencing effect. Obtained results suggest that 20 nm glycogen nanoparticles are optimal for complexation and efficient delivery of siRNA. The chemical composition, surface charge, and size of glycogen-siRNA constructs were finely controlled to minimize interactions with serum proteins which influence the stability and integrity of the glycogen-siRNA constructs. pH-sensitive moieties were introduced within the construct to enhance early endosomal escape. Using single molecule super-resolution microscopy, we demonstrate that the architecture of glycogen-siRNA constructs and the rigidity of the cationic polymer chains are crucial parameters that control the mechanism of endosomal escape driven by the proton sponge effect. The interactions of glycogen-siRNA constructs with immune cells were also investigated, suggesting that glycogen-siRNA constructs may be cleared from the blood stream by mononuclear phagocytic system, but can still successfully deliver the therapeutic cargo.
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ItemProtein adsorption on particles: from particle engineering to understanding biomolecular corona formation( 2017)In biological fluids, biomolecules bind to particles, forming the biomolecular corona. This corona is generally described as a two-component system ‒ the “hard” and the “soft” corona. The adsorbed layer significantly influences the performance of the particles, both in vitro and in vivo. Thus, understanding and characterizing the biomolecular corona is essential. In this work, zwitterionic replica particles, consisting of poly(2-methacryloyloxy-ethyl phosphorylcholine) (PMPC), were synthesized by surface initiated-atom transfer radical polymerization. To understand factors that influence corona formation, the “hard” biomolecular corona formation on engineered particles in different biological milieu using microfluidic was examined. Key questions, such as the influence of flow, particle surface properties, incubation media, and incubation time were addressed. The data showed that dynamic incubation led to a more complex biomolecular corona, adsorption was suppressed on zwitterionic surfaces, and was enhanced after particle incubation in human blood, compared to human serum. An experimental approach that allowed for in situ monitoring of the “hard” and the “soft” protein corona was further introduced. The technique combined confocal laser scanning microscopy with microfluidics and allowed the study of the time-evolution of protein corona formation. The results showed that corona formation was kinetically divided into three different phases: Phase 1, proteins irreversibly and directly bound to the particle surface; Phase 2, irreversibly bound proteins interacting with pre-adsorbed proteins; Phase 3, reversibly bound “soft” corona proteins. To subsequently correlate the immune response to engineered particles and their “in situ” formed biomolecular corona, human whole blood assays were performed. Besides the usage of zwitterionic polymers, pre-adsorption of specific proteins to the particle’s surface is another commonly used technique to obtain low-fouling surfaces. To understand which parameters exactly, (I) the particle’s surface chemistry, (II) pre-adsorbed single proteins, or (III) the “in situ” formed biomolecular corona from human blood, predominantly trigger association with phagocytic cells, mass spectrometry analysis was combined with human whole blood assays. Mesoporous silica (MS) and zwitterionic PMPC particles were pre-coated with human serum albumin, immunoglobulin G, and complement protein C1q. Human whole blood assays revealed that cell–particle interactions were mainly dominated by the respective original surface chemistries of the particles, and less by the pre-adsorbed proteins. Furthermore, the compositions of the individual biomolecular coronas strongly differed between MS and PMPC particles, however remained similar for protein pre-coated systems. The presented work, allows understanding the consequences of particle design parameters and biological factors on (1) the corona composition, (2) its dynamic assembly and structural integrity, and (3) interactions with the human blood. The data might assist in the design of advanced drug delivery vehicles with improved circulation half-life due to suppressed protein adsorption.
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ItemAntibacterial, macroporous chitosan hydrogels for soft tissue engineering applications( 2017)One of the goals of tissue engineering is to be able to develop functional tissue substitutes that can one day replace the function of an organ that is damaged or requires replacement. The development of macroporous scaffolds that can potentially replicate or mimic the functionality and architecture of native extracellular matrix (upon implantation at the site of defect) is one of the major focus in the field of tissue engineering. For a large number of tissue engineering strategies surgical implantation of the scaffold is necessary, yet it also increases the risk of infections. The growing of antimicrobial resistance has necessitated the development of new strategies to tackle drug-resistant bacteria. The focus of this study was to develop porous hydrogel scaffolds for soft tissue engineering and antimicrobial wound healing. This research was first focused on the development of macroporous chitosan hydrogel scaffolds utlilising thermally induced phase separation (TIPS). Two different types of gelation (freeze neutralisation vs freeze crosslinking) were employed for the fabrication of porous chitosan scaffolds. Pore size and porosities are important scaffold parameters for neovascularisation, potentially improving the incorporation of the implant into the host tissue. Hence, various physical parameters such as temperature, chitosan concentration and crosslinking were varied to study their effects on the pore size distribution (60-150 µm) and porosity (65-80%) of the chitosan scaffolds. Mechanical properties of these scaffolds were tunable (compressive modulus: 1-450 kPa) via change of crosslinking and chitosan concentrations. The study was also able to demonstrate scaffold mechanical properties which were relatively similar to the that of the soft tissues (2.5-40 kPa). Mouse 3T3 fibroblasts (relevant cell line for dermis) were shown to adhere, proliferate and penetrate into the scaffolds in vitro. A novel combined mechanical gas foaming and TIPS technique was also developed in order to better control the pore size and morphology of the scaffolds. To establish the range where mechanical foaming results in optimal entrainment of air (hence porous voids) the effects of surfactant (polyvinyl alcohol (PVA)) concentration, foaming speed, foaming time and chitosan concentration were probed and correlated to the foam generating potential. Upon establishing the physical constraints on mechanical foaming for this system, various parameters (PVA concentration, chitosan concentration, mixing speed) that could affect the pore size distribution of these chitosan/PVA scaffolds were studied. This combined technique yielded scaffolds with a more homogeneous spherical morphology. Although pore sizes were generally larger (average pore sizes 120-170 µm) compared to TIPS standalone method, however, pore size distributions were wide and highlighted the need for further improvement of pore size distribution. Scaffolds fabricated with this technique presented soft mechanics (elastic modulus: 2.5-25 kPa), which were similar to those of soft tissues. Experiments with mouse 3T3 fibroblasts revealed that these novel scaffolds supported cell proliferation, attachment without any observable cytotoxicity. Wet chemical synthesis was utilised in order to incorporate selenium (Se) and silver (Ag) nanostructures (separately) into the novel foamed TIPS scaffold, to improve its antimicrobial properties. The morphology of the Ag and Se nanostructures formed in situ on chitosan/PVA scaffolds revealed stark differences which were attributed to the interaction between the chitosan backbone and selenite ions via a hydrogen bonding mechanism. This study also demonstrated the fine control of the loading of both the elements and measured its release in various culture media (complete DMEM, LB media and deionised water). There were significant differences in release of Ag species in all three types of media, indicating the possible role of O2, chloride ions and various organic molecules (e.g., glucose, proteins) that can affect Ag release. Se release was largely unaffected in DMEM and LB media. Extracts from chitosan/PVA scaffolds loaded with various doses of Se (0, 0.06, 0.25, 0.92, 3.67 w/w %) or Ag (0, 0.37, 2.33, 8.97, 17.79 w/w %) showed significant toxicity for Ag based scaffolds. However, no significant cytotoxicity was observed for Se-chitosan/PVA scaffolds. Antibacterial studies carried out on S.aureus, MRSA and E.coli via a Syto9/PI flow cytometric assay revealed significant membrane damage in presence of Se scaffolds and no membrane damage for Ag scaffolds. Colony forming unit (CFU) counts of all three bacterial species tested revealed that CS-Ag caused significant cell death to all bacterial species. However, CS-Se did not lead to significant reduction in bacterial cell numbers, which suggested that while CS-Se extracts caused membrane damage, it did not lead to cell death at the loadings tested. Overall, this thesis focuses on the development of soft scaffolds with tunable pore sizes, morphology and porosities utilising TIPS and gas foaming methodologies. The results also indicate these scaffolds have soft mechanics, support mouse 3T3 fibroblast proliferation, attachment without cytotoxicity. This point towards the possible application of these scaffolds for wound healing. Furthermore, incorporation of Se nanostructures led to enhancement of the scaffold’s bacterial cell permeabilisation properties which could be useful as an adjuvant to enhance the potency of current antimicrobials against drug resistant bacteria.
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ItemEngineering biomacromolecule-based particles with tunable functionality in biological systems( 2016)Particles with tailored physicochemical properties have numerous applications in diagnosis, therapy, and management of human diseases. In this context, elucidation of the interactions between biological systems and nanoengineered materials has emerged as an important research discipline, with the ultimate aim of controlling the interactions to achieve desired physiological responses. Biomacromolecules, such as peptides, proteins and polysaccharides, have diverse physiological functions, such as target recognition, signaling, and catalysis, which remain a challenge to mimic by synthetic methods. Biological systems precisely control synthesis, assembly, and disassembly of the biomacromolecules to guide physiological events. Therefore, controlled assembly of biomacromolecules into nano- and microscale particles may offer a promising platform to study bio-nano interactions, and ultimately to engineer functional materials for biomedical applications. However, previous studies have primarily been limited to particles assembled from biomacromolecules with little function. In this thesis, a robust strategy of assembling functional biomacromolecules into particles is developed, through the use of porous particles as sacrificial templates and reversible chemistry integrated into the biomacromolecular network. The advantages of this strategy include simplicity, versatility, tunability of particle morphology, triggered disassembly, and bioactivity that can be triggered in certain biological conditions. Three types of biomacromolecule-based particles were engineered: (1) peptide nanoparticles with proapoptotic activity (Chapter 3), (2) protein particles with pH-triggered recovery of enzymatic activity (Chapter 4), and (3) polysaccharide-based particles that can be targeted to tumour associated macrophages and Escherichia Coli (Chapter 5). These systems are used to demonstrate how the functionality of the particles in biological systems can be tuned using a chemistry and materials science-based approach.
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ItemSupramolecular polymers as building blocks for the formation of particles( 2014)Over the last two decades, there has been a growing interest in the development of supramolecular polymers, linear macromolecules whose monomeric components are held together by non-covalent interactions. Such supramolecular assemblies are commonly found in nature and are crucial for the function of living tissues and cells. The recent development of synthetic supramolecular polymers has shown promise for enhancing the properties of polymeric materials. Indeed, studies have shown that such materials have significant benefits when compared to conventional, covalently bound, polymers. These benefits are due to the ability of supramolecular assemblies to respond to stimulus, and to dynamically rearrange their structure in a manner unachievable using conventional, covalently bound polymers. Resemblances between the dynamics of synthetic supramolecular polymers and naturally occurring supramolecular polymers are suggestive of their potential for biomedical applications. In this trend, the most promising supramolecular polymer, cyclodextrin (CD) based polyrotaxanes (PRXs), is now emerging as a potential tool to synthetically form dynamic interfaces for applications in the biomedical field. The recent popularity of these polymers in this field is not only due to their inherent, non-covalent properties but also to the low cost, high engineerability and low toxicity of the components they are made of. In this work, CD-based PRXs have been used as building blocks to form particles that were designed for developments in drug delivery. Specifically, the properties specific to PRXs have been exploited to design particles with degradation or stimuli-based response. The unique characteristics of PRXs were found to translate into similarly unique characteristics of the assembled particles. Different approaches have been studied and their advantages and limitations are highlighted. Initial investigations were aimed at designing particles fitting the requirements in properties and specific characteristics highlighted by recent in vivo and in vitro studies. In this direction, we demonstrated controlled degradation of self-assembled PRX-based structures through stimuli triggered disassembly. Such control was also shown for PRX particles dynamically formed using a templated approach, for which disassembly through judicious selection of specific building blocks is highlighted. The use of the templated approach was shown to be more straightforward and versatile in its applications, laying out a framework to form and engineer particles using PRXs as a building block. Lastly, by using CD’s molecular mobility in the PRX as an additional handle for tuning; a “one block” polymer, able to reversibly segregate into multi-blocks leading to the formation of nanoparticles, was developed. This approach is particularly interesting as many responsive polymeric materials have their response due to a stretched-to-coiled transition of individual chain while we show here a transition between a mono-block like architecture to a multi-block like architecture. The preliminary results highlight the potential of PRXs as building blocks for applications in drug delivery systems.
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ItemSoft polymeric nanoparticles as additives for CO2 separation membranes( 2014)The use of polymeric membranes for gas separation has experienced a major expansion in the past few decades with current applications which include the separation of CO2 from flue gas. Various approaches have been explored to fabricate membranes with superior separation performance that can exceed the current upper performance limit. These include the incorporation of hard inorganic nanoparticles into polymers to form mixed-matrix membranes (MMMs). The performance of MMMs can be further enhanced if they can be fabricated into asymmetric morphology. The fabrication of asymmetric membranes, in the form of a thin film composite (TFC) membrane, is more commercially viable due to the increased flux and reduced consumption of expensive nanoparticles. TFC membranes are typically composed of a porous support coated with a highly permeable gutter layer, which is in turn coated with a thin active layer. However, the development of effective asymmetric MMMs has been limited. This is due to the difficulty in fabricating nanoparticles in a size that does not exceed the thickness of the active layer and in avoiding defects in the resulting composite structure. The fabrication of next generation mixed-matrix gas separation membranes is also hampered by the need to ensure a defect-free polymer/inorganic particle interface. A similar approach can be applied to the addition of soft polymeric nanoparticles into a selective polymer matrix. In this case, the problem of defects occurring between the particle and the matrix can be avoided through the engineering of particles that are compatible with the polymer matrix. Hence, this thesis aims to synthesize novel soft polymeric nanoparticles with well-defined architectures and utilize these as additives to be incorporated into the thin active layer of TFC membranes. The requirements for these nanoparticles include (a) a soft and CO2 permeable core and (b) a corona which is compatible with the polymer matrix. The best candidate nanoparticles are then blended with a selective polymer matrix to form the active layer of TFC membranes, which are tested for their CO2 separation from N2. The size of the soft polymeric nanoparticles are significantly smaller than the thickness of the active layer and overcome the problem of blending larger inorganic nanoparticles to form asymmetric MMMs. The first soft polymeric nanoparticles studied were based on triblock copolymers containing polyimide (PI) and poly(dimethylsiloxane) (PDMS). Well-defined difunctional PI was initially prepared via step-growth polymerization. Subsequently, PI was functionalized and chain extended with different molar ratios of PDMS-monomethacrylate (PDMS-MA) via atom transfer radical polymerization (ATRP) to form a series of triblock copolymers. Self-assembly of triblock copolymers in a selective solvent for PI, followed by cross-linking via hydrogen abstraction, resulted in the formation of well-defined nanoparticles with a soft PDMS core. The second soft polymeric nanoparticles developed in this study was based on diblock copolymers containing poly(ethylene glycol) (PEG) and PDMS. Commercially available PEG was utilized as a substitute for the PI block due to the difficulty in synthesizing well-defined polymers via step-growth polymerization. Three different molecular weights of monomethyl ether PEG were initially functionalized to form macroinitiators suitable for ATRP. These macroinitiators were then chain extended with PDMS-MA and photoactive anthracene moeities in different molar ratios to afford a series of photoresponsive diblock copolymers. Self-assembly of diblock copolymers in a selective solvent for PEG, followed by photocross-linking via [4+4] photodimerization of anthracene moeities, resulted in the formation of another well-defined soft polymeric nanoparticles with various structures that range from spherical micelles to large compound micelles. The preparation of soft polymeric nanoparticles through the self-assembly of block copolymers is generally carried out in low concentration to avoid aggregation of nanoparticles. This hinders the preparation of nanoparticles on a larger scale. The third soft polymeric nanoparticles explored in this thesis were based on PEG and PEG-b-PDMS grafted star polymers that were synthesized via the ‘core-first’ approach. This method allows the preparation of nanoparticles in high yields as the crude reaction mixture only requires separation from unreacted monomers. Various grafted star polymers with different PEG and PDMS molar ratios were synthesized in high yields and high conversions utilizing a four-arm ATRP initiator. These grafted star polymers were then utilized as additives for existing gas separation membranes. TFC membranes were prepared from commercially available selective poly(amide-b-ether) (Pebax® 2533) that was blended with a series of PEG and PEG-b-PDMS grafted star polymers. These blends formed a thin film on microporous polyacrylonitrile substrates which have been pre-coated with a PDMS gutter layer. Their ability to selectively separate CO2 from N2 was studied at 35°C and an upstream pressure of 3.4 bar. The addition of soft polymeric nanoparticles into the thin active layer of TFC membranes resulted in greatly improved flux as these particles are able to form localized, high flux, soft domains within a selective polymer matrix. These results create an interesting route to further develop and utilize soft polymeric nanoparticles as additives in membranes for gas separation processes.