Nanodiamonds (NDs) are the most biocompatible member of the carbon nanofamily which are widely researched for diagnostic and therapeutic applications. Unlike other carbon nanomaterials, the surface of NDs is innately reactive, hence capable of conjugating various chemical moieties for targeted actions. This work focuses on utilizing the surface reactivity of NDs for gene therapeutics and addressing the challenges associated with its application in the biological environment. Pristine carboxylated NDs were functionalized with basic amino acids (lysine and lysyl-histidine) through covalent conjugation via a three carbon chain linker. Amino acid functionalized NDs were characterized by infrared spectroscopy, thermogravimetry and size and zeta potential measurements. Lysine conjugation was evident through a marked change in the zeta potential of ND dispersion from negative to a high positive value (-54.6 mV to +26.3 mV). The thermogram of lysine functionalized NDs (Lys-NDs) revealed a significant weight loss from 150ᵒC to 700ᵒC confirming the functionalization through loss of amino acid conjugates from the surface and total loading was calculated as 1.97 mmols/g. Lys-NDs also showed optimum binding with pDNA and siRNA at weight ratios of 1:1 and 1:20 (pDNA/siRNA:ND), respectively. Functionalization of NDs with lysine contributed to limiting aggregation and enhancing the colloidal stability of ND dispersions in biological milieu. The aqueous dispersion of lys-NDs showed minimum sedimentation and remained stable over a period of 25 days. Average sizes under 100 nm and zeta potentials higher than +20 mV indicate a preservation of the cationic surface throughout the testing period. Moreover, size distributions and zeta potentials changed significantly upon incubation of lys-NDs with blood serum suggesting an interaction with biomolecules, mainly proteins and a possible formation of a protein corona. Cellular internalization of bare lys-NDs and their diamoplexes (i.e. complexes of NDs with nucleic acids) was assessed through scanning transmission X-ray microscopy and flow cytometry. Functional efficiency of lysine NDs was determined by flow cytometry monitoring the GFP knockdown through anti-GFP siRNA delivery. Results reveal a promising GFP knockdown of ~17% upon treating the cells with NDs/siRNA diamoplexes at a ratio of 20:1. Subsequent analyses regarding the effect of NDs to prevent cellular proliferation and to cause cellular apoptosis confirmed that they are innately biocompatible at a wide range of concentrations. Unlike lysine NDs, lysyl-histidine functionalization was limited and the surface loading of this conjugate on NDs was very low. Therefore, they were unable to bind pDNA and siRNA even at high weight ratios and hence demand design modifications. Overall this work demonstrates a novel approach of functionalizing NDs with basic amino acids capable of enhancing colloidal stability and delivering of therapeutic genes into mammalian cells. It represents an important step in the development of safe and efficient gene therapy for inherited and acquired diseases.

Liposomes have established roles in drug delivery and cell membrane studies. Amongst other applications; they can also be used as analytical reagents, particularly in immunoassays. Liposomal immunoassays have potential advantages over alternatives; including sensitivity, speed, simplicity and relative reagent stability. The aim of these studies was to develop and evaluate novel examples of these assays. When liposomes entrapped the dye, Sulphorhodamine B, a shift in its maximum absorption wavelength compared to free dye was observed. This was attributed to dimerization of the dye at high concentrations. If the liposomes were disrupted, the released dye was diluted into the external buffer, and the dye's absorption spectrum reverted to that of free dye. After optimization of dye entrapment, immunoassays were developed using these liposomes. Albumin-coated liposomes were used in a model assay to measure serum albumin. This assay employed complement-mediated immunolysis, commonly used in liposomal immunoassays. The liposomes were lysed by anti-albumin and complement, and this could be competitively inhibited by serum albumin. To improve sensitivity, Fab' anti-albumin liposomes were prepared. These enabled measurement of urinary albumin by a complement-mediated immunoassay, but using a sandwich technique. Anti-albumin (intact) liposomes were shown to precipitate on gentle centrifugation after reaction with albumin. They were applied as a solid phase reagent in an heterogeneous immunoassay, using radioimmunoassay for urinary microalbumin as a model assay. Liposomes containing Sulphorhodamine B were also used in a more novel assay; for serum anticardiolipin antibodies. Cardiolipin-containing liposomes were prepared. These were lysable using magnesium ions. Anticardiolipin antibodies (IgG) were found to augment this lysis, enabling their estimation. Similar imprecision and acceptable correlation with a commercial enzyme-linked immunosorbent assay (ELISA) were obtained. The findings demonstrate Sulphorhodamine B release can be used as a marker in homogeneous colorimetric liposomal immunoassays; both in model assays and in potentially more useful clinical biochemistry applications.

Metal-polymer nanocomposites consisting of gold nanorods and temperature-responsive hydrogel nanoparticulates were investigated for use in externally-controlled drug delivery systems. Several different thermo-responsive hydrogels including poly(N-isopropyl acrylamide) (PNIPAAm) and poly(N-isopropryl acrylamide-co-acrylic acid) (P(NIPAAm-co-AA)) nanoparticles were synthesized for these nanocomposites using an aqueous dispersion polymerization method. In addition, nanoparticles of interpenetrating polymer networks (IPN) composed of poly(acrylamide) (PAAm) and poly(acrylic acid) (PAA) were synthesized using a water-in-oil emulsion polymerization. Temperature-responsive equilibrium swelling behavior of nanoparticles with varying crosslinking densities was characterized using dynamic light scattering. IPN systems exhibited a positive swelling response upon heating while PNIPAAm and copolymer systems collapsed upon increase in temperature above the transition point. Nanoparticles were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) which demonstrated shape and morphology of polymer particles. Gold-polymer nanocomposites were formed by grafting gold nanorods to the surface of the polymer nanoparticles. Amine-functionalized gold nanorods were coupled to polymers using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) to activate carboxyl groups on the surface of the polymer nanoparticles. TEM confirmed successful formation of the metal-polymer nanocomposites. Loading and release of a model therapeutic were done to assess the potential use of the polymer component of the nanocomposite for drug delivery. Fluorescein, a model for chemotherapeutics, was loaded into P(NIPAAm-co-AA) polymer nanoparticulates. Loading of the compound was shown to be a function of crosslinking density in the polymer network. Maximum loading was achieved using nanoparticles synthesized with a 10 mol% crosslinker feed ratio with entrapment efficiencies of 80.0 % and loading capacities of 12.0 %. Cytotoxicity studies were performed using a NIH/3T3 mouse fibroblast cell model. Cell viabilities in presence of P(NIPAAm-co-AA) nanoparticles were comparable to (not statistically different than) controls at concentrations up to 4 mg/ml. Similarly, gold-polymer composite concentrations up to 0.5 mg/ml caused limited cell death. / text

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The goal of this study was to develop a model of the gastrointestinal tract (GIT) to be used for in vitro testing of oral insulin delivery devices. The method and intensity of mixing and effects of gastrointestinal fluids with and without enzymes were evaluated. Comparisons were made between an actively mixed simulator and a passively mixed simulator, where the actively mixed simulator is a magnetically stirred flask while the passively mixed simulator is a flexible container on a rocking stage. Slower mixing times and larger time constants for mixing were seen for the passively mixed simulator during a pH tracer experiment. Release studies were performed with several oral insulin delivery device models to evaluate the effects of different mixing techniques on insulin release. In all cases, the more intense mixing of the actively mixed simulator resulted in more insulin release. When using a nanoparticle model in intestinal fluid for example, 100% insulin release was observed in the actively mixed simulator while only 53% was released in the passively mixed simulator after 1 hour. Trypsin and pepsin were used to determine the ability of a drug delivery device to protect insulin from enzymatic degradation in which trypsin was added to simulated intestinal fluid and pepsin was added to simulated gastric fluid. Premature insulin release and insulin denaturation at body temperature occurred in the intestinal fluid so the protective effects against trypsin were unable to be effectively evaluated. An increase in insulin loss from 70% to 95% was detected in the presence of pepsin compared to gastric fluid without enzymes in the actively mixed simulator, indicating that acid hydrolysis of insulin as well as protease attack by pepsin will impact the behavior of an insulin delivery device. An improvement in insulin retention was observed in the passively mixed simulator. After 1 hour, insulin retained was increased from 4% in the actively mixed simulator to 10% in the passively mixed simulator, and after 2 hours, this increase was 2% to 7%. Premature insulin release from the delivery device, acid hydrolysis, temperature denaturation, and enzymatic degradation are factors limiting the effectiveness of oral insulin. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-08-19 19:29:52.804

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Several current social agendas in the province of Manitoba are impacting the demographics in our classrooms. Recent changes to the Public Schools Act, the Educational Administration Act, an aggressive immigration initiative and a trend for movement from rural to urban settings have contributed to classrooms where the learning and behaving needs of students are diverse, their motivations to learn are different and they have varied areas of strength and weakness. In order to meet these new challenges and to provide educational programming that reflects the needs of our global society, schools need to change the way they are delivering service to students in schools. This study used qualitative research methods to examine the conditions that facilitate building an inclusive middle school in Manitoba. Data from both focus group interviews and a document study yielded multiple themes under the headings of actions, culture and strategies. A historical scan of the school’s reform process highlighting the changes in beliefs, leadership, structures and processes over time resulted from the data analysis. Collectively, the findings identify the specific steps the school took to move to inclusion. The information contained in this study will provide a path to help educators move to creating inclusive school environments where all students feel a sense of belonging and fulfillment from their educational experiences.