This research successfully created an ultra-small orifice utilizing undercut fabrication process in a droplet-based microfluidics chip. The proposed novel T-junction structure with ultra-small orifice has a lot of advantages, including long-term stability for uniform droplets formation, reproducible ultra-small size droplet and tunable droplet size. The hydraulic diameter of the orifice is under 2 £gm, and the size of micro droplet produced from the orifice can be tuned to less than 10 £gm in diameter. Chitosan droplet can be produced by the proposed chip, which is usually adopted for medical applications. Surface modification technique was applied to modify the surface of microchannel to be hydrophobic for eaily producing hydro-droplets. Experimental results show that the ultra-small orifice microfluidics chip can steadily produce water-in-oil droplets only by controlling the flow ratio between dispersed phase and continuous phase flow rates. The size of the water-in-oil droplets can be tunable from 22 £gm to 6.5 £gm in diameter by adjusting the flow rate ratio of the continuous and disperse phase flows from 1 to 3.5 and the hydraulic diameter of the orifice is 1.1 £gm. And the size of the chitosan-in-oil droplets also can be tunable from 59 £gm to 27 £gm by adjusting the flow rate ratio of the continuous and disperse phase flows from 4 to 8. The proposed microchip has advantages including ease of control, low cost, and high throughput. The proposed technique can be widely applied on emulsion and micro droplet generation.

Recently, there has been increased effort in researching methods for producing hollow nanostructures because of their potential impact in the fields of catalysis, separation processes, drug delivery, and energy storage and conversion devices. The purpose of this thesis is to describe a method for forming hollow inorganic-organic hybrid nanoboxes and nanoframes. This approach relies upon ionic liquid (1-butyl-3-methyl-imidazolium chloride) mediated auto-templating assembly of CaCO3 and chitosan to form nanoframes (two open faces) and nanoboxes (one open face). The average dimension of the nanostructures formed was 339 ± 95 x 299 ± 89 nm. Detailed structure of nanoboxes and nanoframes were obtained by 3-D electron tomography and X-ray diffraction. Chemical bonding was determined by FTIR, and the ratio of organics to inorganics in the nanostructures was determined by thermal gravimetric analysis. The chitosan to CaCO3 weigh ratio, mixing strength, temperature, and dialysis time were varied to further elucidate the method of formation. It was found that increasing the mixing power caused the equilibrium nanostructure dimension to decrease. On the other hand, varying the experimental temperature in the range of 80 to 160˚C did not affect the nanostructure dimension. The dialysis study showed that during dialysis the nanostructure core was increasingly removed. Nanoframes were observed after 72 hours of dialysis. With further dialysis, there was continued erosion of nanoframes. Results indicate that the concentration gradient and the solubility difference between the mixture components were responsible for this transformation.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2009. / Title from title screen (site viewed January 5, 2010). PDF text: xiv, 328 p. : ill. (some col.) ; 7.48 Mb. UMI publication number: AAT 3373081. Includes bibliographical references. Also available in microfilm and microfiche formats.

Osteoporotic fractures are still the major health concerns in many developed societies especially when the incidence of that tremendously increased with the aging population. However, the outcomes of osteoporotic fracture treatment have not been entirely satisfactory due to the poor quality of bone substance. Inspiringly, bone morphogenetic protein 2 (BMP2) with the ability to accelerate bone formation showed advantages over the conventional treatment. The only problem needed to overcome is its short half-life which resulted in the requirement of readministration and extremely high cost. As a solution to that, gene therapy provides a promising way to sustainably release this protein at the regeneration site. Since viral vectors have been hampered by genetic toxicity and immunogenicity, nanoscaled non-viral vectors offer an attractive means for gene delivery. Chitosan as non-viral vector has been widely investigated for its excellent biocompatibility. Most efforts have been given to improve its low transfection efficiency. In this study, chitosan was first modified with octaarginine, one of cell membrane penetrating peptides, and showed enhanced transfection activity, but which was not significant as expected. Following that, low molecular weight polyethyleminine (PEI) was introduced to modify chitosan through bioreducible disulfide linkage, denoted as Chitosan-ss-PEI. PEI is an efficient non-viral vector but hampered by molecular-weight dependent toxicity. The developed Chitosan-ss-PEI showed good biocompatibility in MTT assay in three different cell lines, during which cells were maintained 80% of viability when the concentration of this vector was up to 100 μg/mL. The optimal transfection efficiency of Chitosan-ss-PEI was higher than that of PEI 25k and comparable to Lipofectamine in delivering luciferase reporter gene. GFP expression mediated by Chitosan-ss-PEI also showed similar results. Chitosan-ss-PEI was then applied to deliver BMP2 gene to skeletal system cells and exhibited the osteogenic ability. For C2C12 myoblast cells, this system inhibited their myoblast differentiation and induced the osteogenic differentiation. It also showed stronger effect in promoting the differentiation of immature osteblast-like MG63 cells and in inducing C3H10T1/2 mesenchymal stem cells osteogenic differentiation in term of ALP activity and mineralization ability compared with other commercial available non-viral vectors. Primary MSCs such as bone marrow stromal cells (BMSC) and human umbilical cord blood mesenchymal stem cells (hUCB-MSC), are usually more difficult to transfect, but they showed stronger osteogenic differentiation ability induced by this system comparing with the cell lines. BMP2 usually requires extremely high concentration to realize its function. Through the investigation of BMP2 signaling regulation in this study, it was found that parathyroid hormone (PTH) could increase the access of BMP2 ligands to their receptors by negatively influencing BMPs antagonist network, resulted in enhanced BMP2 activity in bone remodeling and in promoting the commitment of MSC to osteoblast lineage both in vitro and in vivo. This course involved the endocytosis of PTHR with a complex of LRP6, which organized antagonist network on the cell surface to shield the BMPs receptors. Novel approaches are expected to be developed based on this mechanism with the purpose of intensifying the therapeutic effect of BMPs. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Due to recent legislation requiring the determining of the pharmacokinetic effect of both enantiomers separately, of any new racemic drug before commercialisation, much research is done to improve and optimise methods for obtaining chirally pure compounds important for the pharmaceutical industry, for example epoxide precursors. To date most experiments regarding the biocatalytic chiral separation of 1,2-epoxyoctane has been done in batch processes. The aim of this study was to optimise the enantioselective hydrolysis of 1,2-epoxyoctane by Rhodosporidiurn tondoides in both a batch and continuous process. The batch process was optimised in terms of stir speed, biomass (cell) concentration and reaction time, while the flow-through reactor (continuous process) was optimised with regards to the flow rate as a function of the pressure and the amount of chitosan and biomass in the reactor. Initial inconsistencies of epoxide concentrations in preliminary studies were found to be due to adsorption by reaction and sampling vessels, and the lower than expected solubility of 1,2- epoxyoctane (3.85 mM instead of 6 mM as reported by previous investigators). The results from the batch process suggest that as the reaction time increases, the % ee-epox increases initially, but decreases after 40 minutes. Optimum yield in terms of % ee-epox were obtained at medium stir speed (400 rpm) and biomass (cell) concentration (13 %). Below these values the % ee-epox increases with an increase in stir speed and/or biomass concentration. Above these values however, the increased stir speed and/or biomass concentration causes abrasion between cells, which negatively affects the % ee-epox. The % ee-diol reached a steady state after 10 minutes, and the effect of the different operating conditions on % ee-diol was negligible. In the flow-through reactor chitosan was used as a spacer material (antifouling agent) to help decrease the fouling due to biomass deposition. The use of chitosan as a spacer ensured higher and stabilised flow rates for extended periods of time. In initial studies 0.5 g chitosan increased the flow rate by 34 % with a resistance removal of 25 %. For 1 g chitosan these values were 130 % flow increase and 57 % resistance removal. The flow rate was optimised in relation to the chitosan amount, biomass (cell) amount and pressure. The maximum flow rate was obtained at a pressure of 40 kPa, using the minimum amount of cells (0.4 g) and a maximum amount of chitosan (1.6 g) / Thesis (M.Sc.)--North-West University, Potchefstroom Campus, 2004.

Chitin, chitosan recovered from fly crabs shells have been investigated by the elemental analysis, potentiometric titraton and FT–IR spectrometry methods. The molecular weght of chitosan was determined by measuring their viscosity. The adsorption kinetics of reactive dye and equilibrium conditions has been investigated. The adsorption of reactive dye on chitin and chitosan proceeds according to pseudo – second – order kinetic equation. Adsorption investigations under equilibrium conditions showed that. Theses results were fitted by both Langmuir and Freudlich models.

This study evaluated the ability of photodynamic therapy (PDT), chitosan nanoparticles (CSnp), or their combination, to inhibit bacterial collagenase-mediated degradation of collagen. Rat type 1 fibrillar collagen matrices were untreated or treated with 2.5% glutaraldehyde (GD), 2.5% GD followed by 1% CSnp, 1% CSnp, PDT, or 1% CSnp followed by PDT. Samples, except untreated controls, were exposed to Clostridium histolyticum collagenase. The soluble digestion products were assessed by hydroxyproline assay and the remaining adherent collagen was quantified by picrosirius red (PSR) staining. Collagen treated with CSnp, PDT, or a combination of CSnp and PDT, exhibited less degradation than controls. The abundance of posttreatment residual collagen correlated with the extent of degradation. Fourier transform infrared (FTIR) spectroscopy analysis showed that PDT treatment enhanced collagen cross-linking. Immunoblotting of sedimented CSnp indicated that CSnp and collagenase bound with low affinity. However, CSnp-bound collagenase showed a significant reduction in collagenolytic activity compared with controls.

This study evaluated the ability of photodynamic therapy (PDT), chitosan nanoparticles (CSnp), or their combination, to inhibit bacterial collagenase-mediated degradation of collagen. Rat type 1 fibrillar collagen matrices were untreated or treated with 2.5% glutaraldehyde (GD), 2.5% GD followed by 1% CSnp, 1% CSnp, PDT, or 1% CSnp followed by PDT. Samples, except untreated controls, were exposed to Clostridium histolyticum collagenase. The soluble digestion products were assessed by hydroxyproline assay and the remaining adherent collagen was quantified by picrosirius red (PSR) staining. Collagen treated with CSnp, PDT, or a combination of CSnp and PDT, exhibited less degradation than controls. The abundance of posttreatment residual collagen correlated with the extent of degradation. Fourier transform infrared (FTIR) spectroscopy analysis showed that PDT treatment enhanced collagen cross-linking. Immunoblotting of sedimented CSnp indicated that CSnp and collagenase bound with low affinity. However, CSnp-bound collagenase showed a significant reduction in collagenolytic activity compared with controls.

Due to recent legislation requiring the determining of the pharmacokinetic effect of both enantiomers separately, of any new racemic drug before commercialisation, much research is done to improve and optimise methods for obtaining chirally pure compounds important for the pharmaceutical industry, for example epoxide precursors. To date most experiments regarding the biocatalytic chiral separation of 1,2-epoxyoctane has been done in batch processes. The aim of this study was to optimise the enantioselective hydrolysis of 1,2-epoxyoctane by Rhodosporidiurn tondoides in both a batch and continuous process. The batch process was optimised in terms of stir speed, biomass (cell) concentration and reaction time, while the flow-through reactor (continuous process) was optimised with regards to the flow rate as a function of the pressure and the amount of chitosan and biomass in the reactor. Initial inconsistencies of epoxide concentrations in preliminary studies were found to be due to adsorption by reaction and sampling vessels, and the lower than expected solubility of 1,2- epoxyoctane (3.85 mM instead of 6 mM as reported by previous investigators). The results from the batch process suggest that as the reaction time increases, the % ee-epox increases initially, but decreases after 40 minutes. Optimum yield in terms of % ee-epox were obtained at medium stir speed (400 rpm) and biomass (cell) concentration (13 %). Below these values the % ee-epox increases with an increase in stir speed and/or biomass concentration. Above these values however, the increased stir speed and/or biomass concentration causes abrasion between cells, which negatively affects the % ee-epox. The % ee-diol reached a steady state after 10 minutes, and the effect of the different operating conditions on % ee-diol was negligible. In the flow-through reactor chitosan was used as a spacer material (antifouling agent) to help decrease the fouling due to biomass deposition. The use of chitosan as a spacer ensured higher and stabilised flow rates for extended periods of time. In initial studies 0.5 g chitosan increased the flow rate by 34 % with a resistance removal of 25 %. For 1 g chitosan these values were 130 % flow increase and 57 % resistance removal. The flow rate was optimised in relation to the chitosan amount, biomass (cell) amount and pressure. The maximum flow rate was obtained at a pressure of 40 kPa, using the minimum amount of cells (0.4 g) and a maximum amount of chitosan (1.6 g) / Thesis (M.Sc.)--North-West University, Potchefstroom Campus, 2004.

In some diseases it is preferable that the drugs used in their treatment are released in the colon. The colon is also suitable for systemic delivery of a variety of drugs. A variety of systems have been developed for the purpose of achieving colonic targeting. These approaches are either drug-specific (prodrugs) or formulation specific (coated or matrix preparations) and depends on the pH, transit time and pressure or bacteria in the colon. Different polymers, like chitosan, have been evaluated for their susceptibility to degradation by these bacterial enzymes. Chitosan is considered a good candidate for bacterial degradation and is widely available at low cost and has favourable biological properties. To investigate the influence of formulation factors on the properties of chitosan minitablets, it was necessary to ensure that the chitosan had satisfactory powder flow characteristics to ensure uniform compression in the tablet press and to prevent unacceptable variation in the tablet properties such as weight, thickness, disintegration and strength. Moisture content of the powder, particle size and the inclusion of glidants had an effect on the flowability and it could be improved from a composite flow index value of 32.7 to a value of 58.8. The compressibility of chitosan is very poor and different factors that might influence it, was investigated. Compression forces of between 15 and 20 bar resulted in tablets with acceptable physical characteristics. An increase in moisture content, using the powder fraction > 212 ym as well as a decrease in powder weight resulted in tablets with a higher tensile strength. Lower compression forces resulted in tablets that are extremely porous. This suggests that the chitosan can only be compressed at high compression forces which are difficult to obtain using a standard tablet press. The standard tablet press was therefore modified to fill more powder in the die and generate higher compression forces. Minitablets were compressed and the dissolution of isoniazide from these tablets was investigated. Varying the punch depth or the compaction of the powder did not result in the desired slower release of the drug as a result. The porosity of the tablets compressed at all the punch depth settings and compaction percentages was probably too high to have an effect on the wettablity of the tablets and as a result on the dissolution of the isoniazide from the tablets. The inclusion of excipients such as citric acid (an organic acid which would lower the pH in the tablet, allowing the chitosan to form a gel) and pectin (which would form an insoluble complex with the chitosan) into the formulation delayed the dissolution of the isoniazide from the minitablets. Coating of the minitablets with an enteric coating (Eudragit S ®) initially delayed the dissolution of the isoniazide and would protect the tablets from the harsh environment of the stomach so that the tablets will reach the colon and release the drug. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007