Intracellular delivery of macromolecules is crucial for the success of many research and clinical applications. Several conventional intracellular delivery methods have been used for many years but are still inadequate for several applications because of the issues associated with toxicity, low-throughput, and/or difficulty to target certain cell types. In this study, we developed and evaluated new high-throughput intracellular delivery methods for the efficient delivery of macromolecules while maintaining high cell viability. First, we studied the feasibility of using an array of nanoneedles, with sharp tip diameters in the range of tens of nanometers, to physically make transient holes in cell membranes for intracellular delivery. Puncture loading and centrifuge loading methods were developed and assessed for the effect of various experimental parameters on cell viability and delivery efficiency of fluorescent molecules. In both methods, high-throughput intracellular delivery was feasible by creating transient holes in cell membranes with the sharp tips of the nanoneedles. The second physical intracellular delivery method we studied was a novel microfluidic device that created transient holes in the cell membrane by mechanical deformation and shear stress to the cell. We observed efficient delivery of fluorescent molecules and studied the effect of device design and flow pressure on the delivery efficiency compared to data in the literature. We accounted for cell loss and clogging in the microfluidic devices and determined the true loss of cell viability associated with this method. Lastly, we investigated the possibility of intracellular delivery using nanoparticles on a leukemia cell line. Among number of materials for nanoparticles tested, mesoporous silica/poly-L-lysine nanoparticles were selected for further intracellular delivery study based on cell viability and intracellular delivery capability. We demonstrated the co-delivery of protein and plasmid by encapsulating into and coating onto the surface of the nanoparticles, respectively, which would be advantageous for certain therapeutic strategies. In summary, this work introduced two new intracellular delivery methods involving nanoneedles and novel nanoparticles, and provided an early, independent assessment of microfluidic delivery, showing the strengths and weaknesses of each method. These methods can be further optimized for a number of laboratory and clinical applications with continued research.

An essential component of developing successful neural stem cell (NSC)-based therapies involves the establishment of methodologies to noninvasively monitor grafted NSCs within brain tissues in real time. In this context, ex vivo labeling with ultrasmall superparamagnetic iron oxide (USPIO) particles has been shown to enable efficient tracking of transplanted NSCs via magnetic resonance imaging (MRI). However, whether and how USPIO labeling affects the intrinsic biology of NSCs is not thoroughly understood, and remains an active area of investigation. Here, we perform a comprehensive examination of rat NSC survival and regenerative function upon labeling with the USPIO, Molday ION Rhodamine B (MIRB), which allows for dual magnetic resonance and optical imaging. After optimization of labeling efficiency, two specific doses of MIRB (20 and 50 mu g/mL) were chosen and were followed for the rest of the study. We observed that both MIRB doses supported the robust detection of NSCs, over an extended period of time in vitro and in vivo after transplantation into the striata of host rats, using MRI and post hoc fluorescence imaging. Both in culture and after neural transplantation, the higher 50 mu g/mL MIRB dose significantly reduced the survival, proliferation, and differentiation rate of the NSCs. Interestingly, although the lower 20 mu g/mL MIRB labeling did not produce overtly negative effects, it increased the proliferation and glial differentiation of the NSCs. Additionally, application of this dose also changed the morphological characteristics of neurons and glia produced after NSC differentiation. Importantly, the transplantation of NSCs labeled with either of the two MIRB doses upregulated the immune response in recipient animals. In particular, in animals receiving the 50 mu g/mL MIRB-labeled NSCs, this immune response consisted of an increased number of CD68(+)-activated microglia, which appeared to have phagocytosed MIRB particles and cells contributing to an exaggerated MRI signal dropout in the animals. Overall, these results indicate that although USPIO particles, such as MIRB, may have advantageous labeling and magnetic resonance-sensitive features for NSC tracking, a further examination of their effects might be necessary before they can be used in clinical scenarios of cell-based transplantation.

Gold nanoparticles can serve as an ideal radiosensitizer for radiation therapy due to the high-atomic-number nature of gold and the increased tumor specificity in nanoparticle form. The degree of radiosensitization is highly dependent on both the local gold nanoparticle concentration in the tumor and the radiation source type. Previous Monte Carlo simulations have demonstrated that the gamma-ray energy spectrum of Ytterbium-169 is a strong candidate for a high dose rate brachytherapy implementation of gold nanoparticle-aided radiation therapy. Therefore, the current study focuses on the design of a high dose rate Ytterbium-169 source that would maximize dose enhancement during gold nanoparticle-aided radiation therapy; while meeting the practical constraints for the production of a clinically relevant brachytherapy source. Different encapsulation materials are studied in order to determine its effect on the dosimetric characteristics of the source. Specifically, the photon spectra, secondary electron spectra, and dose enhancement characteristics are calculated via Monte Carlo simulations to elucidate the effects on potential radiosensitization during gold nanoparticle-aided radiation therapy. Furthermore, this project involves a study into the modification of external x-ray beams from a Philips RT-250 orthovoltage x-ray machine in an attempt to match the dosimetric characteristics of the Ytterbium-169 brachytherapy source. This investigation will enable the production of an external beam that can serve as a good surrogate of an actual brachytherapy source and facilitate the pre-clinical investigation of gold nanoparticle-aided radiation therapy with Ytterbium-169.

The body’s humoral immune response plays a larger role in the body’s defenses beyond screening for invading pathogens. Modulation of this response is also vital for tissue regeneration, drug delivery, and vaccine development. The immune system operates within a complicated feedback loop and as such, altering the strength of the immune response can be approached from an engineering perspective. While a strong initial input can direct the response to either a pro- or anti-inflammatory bias, extreme responses can be deleterious, as in the case of allergic reactions or sepsis. Therefore, the objective of this thesis was to develop a novel biomaterials platform that can be used to alter the immune response in a tunable manner. Antibodies are not only the workhorses of the adaptive immune response but are also powerful immunomodulators through their Fc (constant fragment) regions. By coating microparticles with Fc ligands in variable surface densities, we were able to utilize the sensitivity of multivalent signaling to tune the response of the immune response. Microparticle size was also varied to decouple the effects of physical versus biochemical signaling. The goal of this thesis was to analyze the effects of Fc coated particles on two major components of the humoral immune responses: macrophages and the complement system. We first looked at the mechanical response of macrophages through phagocytosis and found that both Fc density and microparticle size had significant impacts on macrophage phagocytosis. These results also provide a particle delivery “toolbox” for future applications. We then analyzed the downstream effects of Fc particles on macrophage phenotype and on phenotype plasticity. This showed that the addition of Fc particles lead to increased production of TNFα and IL-12 and inverted the response of LPS treated macrophages. Finally, we applied our particles to activate the complement system, an often overlooked cascade of serum protein activation that results in bacterial cell lysis. Cleaved components of the complement system are also powerful chemokines and can act as a vaccine adjuvant. Fc density on particles played a large role in complement system activation, both through the classical and alternative pathway, as it lead to a binary response for smaller particles and a tunable response for larger particles. We then applied these results to create a novel form of antibiotic by using Fc particles to direct complement-mediated bacterial cytotoxicity. The use of immune activation by Fc particles was also applied to better understand and improve the tuberculosis vaccine. Our findings are significant to the biomaterials and immunology fields as we showed that Fc microparticles can generally be used to alter the immune response in a tunable manner for a broad range of applications, as well answering fundamental immunology questions.

Interstitial carbides and nitrides of early transition metals in Groups IV-VI exhibit platinum-like behavior which makes them a promising candidate to replace noble metals in a wide variety of reactions. Most synthetic methods used to prepare these materials lead to bulk or micron size powder which limits their use in reactions in particular in catalytic applications. Attempts toward the production of transition metal carbide and nitride nanoparticles in a sustainable, simple and cheap manner have been rapidly increasing. In this thesis, a new approach was presented to prepare nano-scale transition metal carbides and nitrides of group IV-VI with a size as small as 3 nm through the reaction of transition metal precursor with mesoporous graphitic carbon nitride (mpg-C3N4) that not only provides confined spaces for nanoparticles formation but also acts as a chemical source of nitrogen and carbon. The produced nanoparticles were characterized by powder X-ray diffraction (XRD), temperature-programmed reaction with mass spectroscopy (MS), CHN elemental analyses, thermogravimetric analyses (TGA), nitrogen sorption, X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). The effects of the reaction temperature, the ratio of the transition metal precursor to the reactive template (mpg-C3N4), and the selection of the carrier gas (Ar, N2, and NH3) on the resultant crystal phases and structures were investigated. The results indicated that different tantalum phases with cubic structure, TaN, Ta2CN, and TaC, can be formed under a flow of nitrogen by changing the reaction temperatures. Two forms of tantalum nitride, namely TaN and Ta3N5, were selectively formed under N2 and NH3 flow, respectively. Significantly, the formation of TaC, Ta2CN, and TaN can be controlled by altering the weight ratio of the C3N4 template relative to the Ta precursor at 1573 K under a flow of nitrogen where high C3N4/Ta precursor ratio generally resulted in high carbide content rather than nitride. In addition, the reactivity of the transition metals of group IV-VI with the reactive template was investigated under a flow of N2 at different temperatures in the range of 1023 to 1573 K while keeping the weight ratio constant at 1:1. The results show that Ti, V, Nb, Ta, and Cr reacted with mpg-C3N4 at 1023 K to form nitride phase with face centered cubic structure. The nitride phase destabilized at higher temperature ≥1223 K through the reaction with the remaining carbon residue originated from the decomposition of the template to form carbonitride and carbide phases. Whereas, Mo and W produce a hexagonal structure of carbide irrespective of the applying reaction temperature. The tendency to form transition metal nitrides and carbides at 1023 K was strongly driven by the free energy of formation. The observed trend indicates that the free energy of formation of nitride is relatively lower for group IV and V transition metals, whereas the carbide phase is thermodynamically more favorable for group VI, in particular for Mo and W. The thermal stability of nitride decreases at high temperature due to the evolution of nitrogen gas. The electrocatalytic activities of the produced nanoparticles were tested for hydrogen evolution reaction in acid media and the results demonstrated that molybdenum carbide nanoparticles exhibited the highest HER current with over potential of 100 mV vs. RHE, among the samples prepared in this study. This result is attributed to the sufficiently small particle size (8 nm on average) and accordingly high surface area (308 m2 g-1). Also, the graphitized carbon layer with a thickness of 1 nm on its surface formed by this synthesis provides excellent electron pathway to the catalyst which will improve the rate of electron transfer reaction.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: ß-Galactosidase of Escherichia coli is the equivalent of lactase in humans and has the ability to bind and hydrolyse lactose. Lactase de ciency is a common phenomenon present in almost 70% of the world's population. This has resulted in greater than before demands on the food processing industry to develop a method that will allow for the hydrolysis of the disaccharide lactose in milk but will also allow for the removal of the remaining active enzyme. In this thesis, a new method, that is bio-speci c and well characterized for the removal of lactose from a lactose containing solution, is described. The E537D mutated version of ß-Galactosidase, which has a much lower activity compared to the wildtype and is able to bio-speci cally bind lactose for longer periods, was covalently immobilised to commercially available magnetic nanoparticles (fl uidMAG-Amine) via two coupling strategies. Glutaraldehyde is a cross-linking agent that reacts with amine groups, while N- (3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) is a coupling agent that activates carboxylic groups. These agents are widely used for the coupling of biomolecules to solid supports. The covalently coupled fluidMAG-E537D ß-Galactosidase particles were characterized regarding retained enzymatic activity and ability to bind and physically remove lactose from a lactose containing solution by applying an external magnetic eld, after lactose binding, to the enzyme-particle complex in solution. Each component aimed at yielding this functionally immobilised enzyme complex was studied and optimized to contribute to the development of this novel technique, which is a ordable and simple, for the removal of lactose from solution for the ultimate production of lactose free milk. Results indicated the glutaraldehyde method of ß-Gal cross-linking to fluidMAG-Amine to be the preferred strategy since it allowed an increased carrier capacity of protein to the particles. The glutaraldehyde cross-linked protein also exhibited a two-fold higher activity than the EDC coupled protein. Furthermore, the glutaraldehyde cross-linked fluidMAG-E537D ß-Gal was able to physically remove 34 % of the lactose from a 0.2 nmol/L lactose in solution. This, therefore, con rmed the potential use of this novel technique in the food processing industry. / AFRIKAANSE OPSOMMING: ß-Galaktosidase vanaf Escherichia coli is dieselfde as laktase in mense en beskik oor die vermoë om laktose te bind en te hidroliseer. 'n Gebrek aan laktase kom algemeen voor en ongeveer 70 % van die wêreldbevolking ly hieraan. Laasgenoemde het daartoe gelei dat daar meer druk as vantevore op die voedselproduksie industrie is om 'n metode te ontwikkel waarmee die hidrolise van die disakkaried laktose in melk moontlik sal wees asook die verwydering van die oorblywende aktiewe ensiem. In hierdie tesis word 'n nuwe metode beskryf wat biospesi ek en goed gekarakteriseer is vir die verwydering van laktose vanuit 'n laktose bevattende oplossing. Die E537D gemuteerde weergawe van ß-Galaktosidase, wat beskik oor 'n baie laer aktiwiteit as die wildetipe asook die vermoë om laktose biospesi ek vir langer periodes te bind, is kovalent geïmmobiliseer op kommersieel beskikbare magnetiese nanopartikels (fluidMAG-Amine) via twee koppelingsstrategieë. Glutaraldehied is 'n kruisbindingsagent wat met amino groepe reageer, terwyl EDC 'n koppelingsagent is wat karboksie groepe aktiveer. Hierdie agente word algemeen gebruik vir die binding van biomolekules aan soliede matrikse. Die kovalent gekoppelde fluidMAG-E537D ß-Galaktosidase partikels is gekarakteriseer met betrekking tot behoue ensimatiese aktiwiteit en vermoë om laktose te bind en sies te verwyder vanuit 'n oplossing wat laktose bevat deur 'n eksterne magneetveld op die ensiem-partikel kompleks in oplossing toe te pas, nadat die binding van laktose plaasgevind het. Elke komponent van hierdie funksioneel geïmmobiliseerde ensiemkomplekse is ondersoek en geoptimaliseer met die doel om by te dra tot die ontwikkeling van 'n nuwe tegniek wat bekostigbaar en eenvoudig is vir die verwydering van laktose vanuit 'n oplossing vir die uiteindelike gebruik in die produksie van laktose-vrye melk. Resultate het getoon dat die glutaraldehied metode van ß-Gal kruisbinding op fluidMAG-Amine verkies word aangesien dit 'n verhoogde draerkapasiteit van proteïene op die partikels moontlik maak. Die glutaraldehied gekoppelde proteïene beskik ook oor twee keer meer aktiwiteit as die EDC gekoppelde proteïene. Die glutaraldehied gekoppelde fluidMAG-E537D ß -Gal kon 34 % van die laktose teenwoordig in 'n 0.2 nmol/L laktose oplossing sies verwyder. Hierdie het dus die potensiële gebruik van hierdie nuwe metode in die voedselproduksie industrie bevestig.

Medical diagnosis is the process of attempting to determine and/or identify a possible disease or disorder. This process is revealed by biomarkers, defined by The Food and Drug Administration (FDA) as “characteristics that are objectively measured and evaluated as indicators of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”. The process of biomarker discovery has been boosted in the last years by proteomics, a research discipline that takes a snapshot of the entire wealth of proteins in an organism/ tissue/ cell/ body fluid. An implementation of the analysis methods can help in isolate proteins present in the low range of concentrations, such as biomarkers very often are. An established biomarker can further be measured with the help of biosensors, devices that can be employed in the point-of care diagnostics. This PhD thesis shows and discusses the results of three projects in the field of protein biomarkers discovery and quantification. The first project exploited proteomics techniques to find relevant protein markers for Intrauterine Growth Restriction (IUGR) in cordonal blood serum (UCS) and amniotic fluid (AF). A 14 proteins in UCS and 11 in AF were successfully identified and found to be differentially expressed. Molecularly Imprinted Polymers (MIPs) directed towards proteins and peptides containing phosphotyrosine were then produced, with the final goal of selectively extracting phosphopeptides from a peptide mixture. An alteration of the phosphorylation pattern is in fact often associated to important diseases such as cancer. The polymers were produced as nanoparticles, that were characterised with Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). A recipe was also tested for binding capacity towards phosphotyrosine. A Surface Plasmon Resonance (SPR) biosensor to quantify hepcidin hormone was finally produced. This is the major subject in iron homeostasis in vertebrates and marker of iron unbalance diseases. A calibration curve was made and affinity/kinetic parameters for the ligand employed were measured.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy