Investigation of properties affecting controlled release of macromolecules from PLGA microspheres

Poly(lactide-co-glycolide), or PLGA, microspheres offer a widely-studied biodegradable option for controlled release of therapeutics. An array of fabrication methodologies have been developed to produce these microspheres with the capacity to encapsulate therapeutics of various types; and produce microspheres of a wide range of sizes for different methods of delivery. The encapsulation, stability, and release profiles of therapeutic release based on physical and thermodynamic properties has also been studied and modeled to an extent. Much research has been devoted to tailoring formulations for improved therapeutic encapsulation and stability as well as selective release profiles. Despite the breadth of available research on PLGA microspheres, further analysis of fundamental principles regarding the microsphere degradation, formation, and therapeutic encapsulation is necessary. This work aims to examine additional fundamental principles related to PLGA microsphere formation and degradation from solvent-evaporation of preformed polymer. In particular, mapping the development of the acidic microenvironment inside the microsphere during degradation and erosion is discussed. Also, the effect of macromolecule size and conformation is examined with respect to microsphere diameter and PLGA molecular weight. Lastly, the effects of mechanical shearing and protein exposure to aqueous media during microsphere formation are examined. In an effort to better understand the acidic microenvironment development across the microsphere diameter, pH sensitive dye conjugated to protein that undergoes conformational change at different acidic pH values was encapsulated in PLGA microspheres of diameters ranging from 40 µm to 80 µm, and used in conjunction with fluorescence resonance energy transfer to measure the radial pH change in the microspheres. Qualitative analysis of confocal micrographs was used to correlate fluorescence intensity with pH value, and obtain the radial pH across the center of the microsphere. Therapeutic encapsulation and release from polymeric microspheres is governed by an interconnected variety of factors, including the therapeutic itself. The globular protein bovine serum albumin, and the elongated and significantly smaller enzyme, lysozyme, were encapsulated in PLGA microspheres ranging from 40 µm to 80 µm in diameter. The initial surface morphology upon microsphere formation, release profiles, and microsphere erosion characteristics were explored in an effort to better understand the effect of protein size, conformation, and known PLGA interaction on the formation and degradation of PLGA microspheres and macromolecule release, with respect to PLGA molecular weight and microsphere diameter. In addition to PLGA behavior and macromolecule behavior, the effect of mechanical stresses during fabrication was examined. Two similar solvent extraction techniques were compared for the fabrication of albumin loaded microspheres. In particular, the homogeneity of the microspheres as well as capacity to retain encapsulated albumin were compared. This preliminary study paves the way for a more rigorous treatment of the effect of mechanical forces present in popular microsphere fabrication. Several factors affecting protein release from PLGA microspheres are examined herein. The technique explored for spatial resolution of the pH inside the microsphere proved mildly effective in producing a reliable method of mapping microsphere pH changes. However, notable trends with respect to microsphere size, PLGA molecular weight, and microsphere porosity were observed. Proposed methods of improving spatial resolution of the acidic microenvironment are also provided. With respect to microsphere formation, studies showed that albumin and lysozyme had little effect on the internal homogeneity of the microsphere. Rather, ionic interactions with PLGA played a more significant role in the encapsulation and release of each macromolecule. Studies also showed that higher instances of mechanical stress led to less homogeneous microspheres with lower protein encapsulation. This suggests that perhaps instead of or in addition to modifying the microsphere formation formulation, the fabrication technique itself should be more closely considered in achieving homogeneous microspheres with desired loading.

Large eddy simulation of shock boundary layer interaction control using micro-vortex generators

The performance of supersonic engine inlets and external aerodynamic surfaces can be critically affected by shock wave / boundary layer interactions (SBLIs), whose severe adverse pressure gradients can cause boundary layer separation. Currently such problems are avoided primarily through the use of boundary layer bleed/suction which can be a source of significant performance degradation. This study investigates a novel type of flow control device called micro-vortex generators (µVGs) which may offer similar control benefits without the bleed penalties. µVGs have the ability to alter the near-wall structure of compressible turbulent boundary layers to provide increased mixing of high speed fluid which improves the boundary layer health when subjected to flow disturbance. Due to their small size,µVGs are embedded in the boundary layer which provide reduced drag compared to the traditional vortex generators while they are cost-effective, physically robust and do not require a power source. To examine the potential of µVGs, a detailed experimental and computational study of micro-ramps in a supersonic boundary layer at Mach 3 subjected to an oblique shock was undertaken. The experiments employed a flat plate boundary layer with an impinging oblique shock with downstream total pressure measurements. The moderate Reynolds number of 3,800 based on displacement thickness allowed the computations to use Large Eddy Simulations without the subgrid stress model (LES-nSGS). The LES predictions indicated that the shock changes the structure of the turbulent eddies and the primary vortices generated from the micro-ramp. Furthermore, they generally reproduced the experimentally obtained mean velocity profiles, unlike similarly-resolved RANS computations. The experiments and the LES results indicate that the micro-ramps, whose height is h≈0.5δ, can significantly reduce boundary layer thickness and improve downstream boundary layer health as measured by the incompressible shape factor, H. Regions directly behind the ramp centerline tended to have increased boundary layer thickness indicating the significant three-dimensionality of the flow field. Compared to baseline sizes, smaller micro-ramps yielded improved total pressure recovery. Moving the smaller ramps closer to the shock interaction also reduced the displacement thickness and the separated area. This effect is attributed to decreased wave drag and the closer proximity of the vortex pairs to the wall. In the second part of the study, various types of µVGs are investigated including micro-ramps and micro-vanes. The results showed that vortices generated from µVGs can partially eliminate shock induced flow separation and can continue to entrain high momentum flux for boundary layer recovery downstream. The micro-ramps resulted in thinner downstream displacement thickness in comparison to the micro-vanes. However, the strength of the streamwise vorticity for the micro-ramps decayed faster due to dissipation especially after the shock interaction. In addition, the close spanwise distance between each vortex for the ramp geometry causes the vortex cores to move upwards from the wall due to induced upwash effects. Micro-vanes, on the other hand, yielded an increased spanwise spacing of the streamwise vortices at the point of formation. This resulted in streamwise vortices staying closer to the wall with less circulation decay, and the reduction in overall flow separation is attributed to these effects. Two hybrid concepts, named “thick-vane” and “split-ramp”, were also studied where the former is a vane with side supports and the latter has a uniform spacing along the centerline of the baseline ramp. These geometries behaved similar to the micro-vanes in terms of the streamwise vorticity and the ability to reduce flow separation, but are more physically robust than the thin vanes. Next, Mach number effect on flow past the micro-ramps (h~0.5δ) are examined in a supersonic boundary layer at M=1.4, 2.2 and 3.0, but with no shock waves present. The LES results indicate that micro-ramps have a greater impact at lower Mach number near the device but its influence decays faster than that for the higher Mach number cases. This may be due to the additional dissipation caused by the primary vortices with smaller effective diameter at the lower Mach number such that their coherency is easily lost causing the streamwise vorticity and the turbulent kinetic energy to decay quickly. The normal distance between the vortex core and the wall had similar growth indicating weak correlation with the Mach number; however, the spanwise distance between the two counter-rotating cores further increases with lower Mach number. Finally, various µVGs which include micro-ramp, split-ramp and a new hybrid concept “ramped-vane” are investigated under normal shock conditions at Mach number of 1.3. In particular, the ramped-vane was studied extensively by varying its size, interior spacing of the device and streamwise position respect to the shock. The ramped-vane provided increased vorticity compared to the micro-ramp and the split-ramp. This significantly reduced the separation length downstream of the device centerline where a larger ramped-vane with increased trailing edge gap yielded a fully attached flow at the centerline of separation region. The results from coarse-resolution LES studies show that the larger ramped-vane provided the most reductions in the turbulent kinetic energy and pressure fluctuation compared to other devices downstream of the shock. Additional benefits include negligible drag while the reductions in displacement thickness and shape factor were seen compared to other devices. Increased wall shear stress and pressure recovery were found with the larger ramped-vane in the baseline resolution LES studies which also gave decreased amplitudes of the pressure fluctuations downstream of the shock.

The impacts of environmental and anthropogenic stressors on largemouth bass: an integration of field and laboratory studies

Several environmental stressors can impact the physiology and survival of fishes. Fish experience natural fluctuations in temperature and dissolved oxygen, but variations in these parameters due to anthropogenic sources are typically greater in magnitude and duration. Changes in temperature and oxygen of anthropogenic origins may therefore have larger negative impacts on fish than those occurring during natural events. Physiological parameters are sensitive indicators of the impacts of stressors by providing insight into the manner in which fish are disturbed by the stressor. Fish may display cumulative physiological responses to successive stressors, but the concept of synergy among multiple thermal stressors is poorly understood. Further, some fish species can be subjected to competitive angling events, which expose fish to an array of additional stressors that can increase mortality. The impacts of these events may change over seasons as fish display seasonal changes in behavior and physiology. Latitudinal origin may also affect the physiological response and mortality of fish exposed to common environmental stressors as individual populations are adapted to local environmental conditions. This thesis focuses on addressing these potential impacts on physiological parameters and mortality of largemouth bass (Micropterus salmoides) and provides implications for management and conservation. Largemouth bass were relatively robust to abrupt changes in temperature and oxygen, but were perturbed from physiological homeostasis during large (12°C) temperature shocks and low (< 4 mg O2/L) levels of dissolved oxygen. Cumulative physiological impacts of multiple cold shocks were only slightly greater than the disturbances sustained during a single cold shock, suggesting largemouth bass are able to tolerate successive thermal stressors. Largemouth bass exhibited seasonal changes in physiological parameters but the responses of fish to angling tournaments were relatively similar across seasons when compared with seasonal controls. Mortality was low during angling tournaments held during four seasons and no apparent seasonal trends were observed. Lastly, largemouth bass from two latitudinally separated populations exhibited differences in their physiological responses to acute cold stressors and overwinter mortality, characterized by greater mortality and physiological disturbances of southern fish than northern fish. Knowledge gained from this study can be used to make management and conservation decisions regarding a host of environmental factors and provides insight into the mechanisms by which fish species can persist over large latitudinal ranges.

Functional studies of lignin biosynthesis genes and putative flowering gene in Miscanthus x giganteus and studies on indolyl glucosinolate biosynthesis and translocation in Brassica oleracea

The first topic area of this thesis involved studies on the accumulation and translocation of glucosinolates (GSs), bioactive secondary plant compounds, in broccoli plants. Changes in GS accumulation and gene expression levels in response to exogeneous methyl jasmonate (MeJA) treatment were analyzed in different tissue types at different developmental stages of broccoli. Greater accumulation of GSs with MeJA treatment was observed in apical leaves of broccoli seedlings and florets of plants at harvest maturity. Increases in indolyl GS in apical leaves of seedlings and florets were coupled with the up-regulation of indolyl GS biosynthesis genes. The accumulation of indolyl GSs appears to be modulated by MeJA treatment in an organ-specific manner for optimal distribution of defense substances in the plant. Metabolic profiling of hydrophilic metabolites using GC-MS demonstrated increased accumulation of various phenolics, ascorbates and amino acids in broccoli tissues after MeJA treatment. Distinct changes in carbohydrate levels observed between different tissues (vegetative leaves and floret tissues) of broccoli plants after treatment suggest that carbon metabolism is differentially modulated by MeJA treatment in different tissue types depending on sink-source relationships. Reduced levels of hexose sugars and tricarboxylic acid intermediates after MeJA treatment may reflect the increased requirement for carbon and energy needed to drive secondary product biosynthesis to accumulate metabolites for defense against insects and other herbivores. Substantial increases of indolyl and aromatic GSs after exogenous treatment with MeJA in stem and petioles of seedlings and the existence of intact indolyl-GS forms in phloem exudates suggest enhanced de novo synthesis in combination with active transport. Indoly GSs share structural similarities with the auxin, IAA, and may interact with components of the auxin transport system for intra- and extra-cellular transport or translocation. Application of the auxin efflux inhibitor, 1-naphthylphthalamic acid (NPA) reduced MeJA-mediated accumulation of indolyl GSs in broccoli florets and seedling tissues. NPA did not inhibit expression of indolyl GS biosynthesis genes shown to be upregulated by MeJA treatment or the accumulation of tryptophan, the amino acid precursor of indolyl GSs. Exogenous application of benzyl GS to Arabidopsis roots induced ectopic expression of the PIN1 protein associated with the auxin transport system similar to treatment with NPA, again suggesting GS interaction with the auxin efflux carrier system. The inhibitory effect of NPA on MeJA-mediated accumulation of GS may be due to competitive binding of NPA to auxin efflux carrier components and that GS transport is mediated by the auxin transport system. The inhibitory effect of NPA on indolyl and aromatic GS accumulation and the bioactivity of exogenous treatment of these GS compounds in PIN1 localization, Arabidopsis root growth, and gravitrophic response suggest that indolyl and aromatic GSs may be antagonistic to IAA transport and biosynthesis. Indolyl and aromatic GSs can also be potentially converted into IAA by hydrolysis. This intrinsic feature of GSs may be the part of a sophisticated regulatory process where the metabolic pathways in the plant shift from active growth to a reversible defense posture in response to biotic or abiotic stress. It seems likely that indolyl and aromatic GSs are important compounds that provide connections between jasmonate and auxin signaling. Further studies are required to reveal the regulatory mechanism for crosstalk between the two hormones. The third part of this research was to investigate effect of selenium fertilization and MeJA treatment on accumulation of GSs in broccoli florets. Increasing dietary intake of the element selenium (Se) has been shown to reduce the risk of cancer. Simultaneous enhancement of both Se and GS concentrations in broccoli floret tissue were conducted through the combined treatment of MeJA with Se fertilization. A low level of Se fertilization (concentration) with MeJA treatment displayed no significant changes in total aliphatic GS concentrations with 90% and 50% increases in indolyl and total GSs concentrations, respectively. This result suggests that Se- and GS-enriched broccoli with improved health-promoting properties can be generated by this combined treatment. The second topic of this thesis was conducted to provide basic information required to improve biomass quality and productivity and develop tools for gene transformation in Miscanthus x giganteus. The perennial rhizomatous grass, Miscanthus x giganteus is an ideal biomass crop due to its rapid vegetative growth and high biomass yield potential. As a naturally occurring sterile hybrid, M. x giganteus must be propagated vegetatively by mechanicalling divided rhizomes or from micropropagated plantlets. The effect of callus type, age and culture methods on regeneration competence was studied to improve regeneration efficiency and shorten the period of tissue culture in M. x giganteus propagation. Seven lignin biosynthesis genes and one putative flowering gene were isolated from M. x giganteus by PCR reactions using maize othologous sequences. Southern hybridization and nuclear DNA content analysis indicated that the genes isolated from M. x giganteus exist in the genome of other Miscanthus species as multiple copies. Analysis of lignin content and histological staining of lignin deposition indicated that higher lignin content is found in mature stem node tissues compared to young leaves and apical stem nodal tissues. Cell wall lignification is associated with increasing tissue maturity in Miscanthus species. RNAi and antisense constructs harboring sequences of these genes were developed to generate Miscanthus transgenic plants with suppressed of lignin biosynthesis and delayed flowering.