Thesis (Ph.D.) -- University of Texas at Arlington, 2008.

Thesis (M.S.)--University of Akron, Dept. of Biomedical Engineering, 2008. / "August, 2008." Title from electronic thesis title page (viewed 12/9/2009) Advisor, Stephanie T. Lopina; Committee members, Amy Milsted, Daniel B. Sheffer, Daniel Ely; Department Chair, Daniel B. Sheffer; Dean of the College, George K. Haritos; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide, accounting for over 600,000 deaths per year. The most common treatment strategy for intermediate and advanced stage unresectable HCC is transarterial chemoembolization (TACE), which involves the local administration of a chemotherapeutic drug combined with arterial occlusion resulting in ischemic tumor necrosis. However, TACE suffers from inadvertent exposure of noncancerous liver parenchyma to embolic agents resulting in liver injury. In some cases, over-embolization has lead to infection, necrosis of unaffected liver tissue, and even liver failure which suggests the need for a biocompatible, multifunctional embolic material which can deliver anticancer drugs with high target specificity. Our laboratory has recently developed a method to fabricate porous silicon (pSi) microparticles with defined physicochemical properties based on photolithography and anodic etching. These microparticles function as multistage drug delivery systems that can circumvent the biobarriers present in the systemic circulation enabling site-specific localization and release of chemotherapy and imaging agents. The versatility of the fabrication process enables the realization of microparticles ranging in size from 600nm to 116[mu]m in diameter with varying shapes, including discoidal, cylindrical and hemispherical, and varying porosity with pore sizes ranging from 6nm to greater than 50nm in diameter. Nanoparticles, such as quantum dots, siRNA-loaded nanoliposomes, gadolinium-based contrast agents, gold and iron oxide nanoparticles, are loaded in pSi microparticles by tailoring their pore sizes and surface chemistries. This thesis presents preliminary results on the applicability of biocompatible, engineered pSi microparticles as an embolic agent for HCC chemoembolization therapy. Hemispherical microparticles with 116[mu]m diameter were successfully fabricated and suspended in phosphate buffered saline (PBS). A microvascular construct was rapid prototyped in polydimethylsiloxane (PDMS) as an in vitro experimental platform to study the embolization behavior of pSi microparticles. Oxidized pSi microparticles were introduced into the microfluidic device at an appropriate flow rate and time-lapse images were taken showing the formation of occlusions at the bifurcation within minutes of administration. Furthermore, penetration through the bifurcation was completely hindered suggesting that pSi microparticles can potentially be used as a biocompatible, multifunctional chemoembolization agent. Although these results are promising, further investigations are warranted.

The rational and directed delivery of genetic material to the cell is a formidable tool to investigate the phenotypic effects of gene expression regulation and a promising therapeutic strategy for genetic defects. RNA interference constitutes a versatile approach to gene silencing. Despite the development of numerous strategies the transfection of small interfering RNA (siRNA) is highly dependent on cell type and conditions. Direct physical access to the intracellular compartment is a promising path for high efficiency delivery independently of cell type and conditions. Silicon nanowires grant such access with minimal toxic effects, and allow intracellular delivery of DNA when actuated by atomic force microscope. These findings reveal the potential for porous silicon nanostructures to serve as delivery vectors for nucleic acids due to their porous nature, elevated biocompatibility, and biodegradability. This dissertation illustrates the development a novel platform for efficient siRNA transfection based on an array of porous silicon nanoneedles. The synthesis of biodegradable and biocompatible porous nanowires was accomplished by a novel strategy for electroless etch of silicon that allows anisotropic etch simultaneously with porosification. An ordered array of cone shaped porous silicon nanoneedles with tunable tip size, array density and aspect ratio was obtained coupling this strategy with patterned metal deposition and selective reactive ion etch. This process also granted control over porosity, nanopore size and flexural modulus. The combination of these parameters was appropriately optimized to ensure cell penetration, maximize siRNA loading and minimize cytotoxic effects. Loading of the negatively charged siRNA molecules was optimized by applying an external electric field to the nanoneedles under appropriate voltage conditions to obtain a tenfold increase over open circuit loading, and efficient penetration of the siRNA within the porous volume of the needles. Alternative surface chemistry modification provided a means for effective siRNA loading and sustained release. siRNA transfection was achieved by either imprinting the nanoneedles array chip over a culture of MDA-MB-231 cells or allowing the cells to self-impale over the needles. The procedures allowed the needles to penetrate across the cell membrane without influencing cell proliferation. siRNA was successfully transfected and was effective at suppressing gene expression. / text

Nanoparticles have garnered much attention in pharmaceutical and biomedical fields because their small size and high surface area facilitate drug absorption, improve access to cells and organs, and enhance optical imaging. However, delivery of nanoparticles to the body is not always feasible or effective. Here, nanoparticle assemblies (flocs or clusters) for pulmonary drug delivery and biomedical imaging in cells are shown to facilitate delivery, interactions with cells, and manipulation of optical properties of inorganic/organic nanocomposites. The formation of aggregates by physical techniques and their mechanisms are described in detail. For pulmonary delivery, particles with aerodynamic diameters between 1-5 [mu]m deposit efficiently in the deep lungs. However, crystalline, non-porous, poorly water soluble drugs of this size require long dissolution times, limiting absorption by the body. Therefore, drug dissolution must be “decoupled” from deposition to improve absorption. To address this challenge, drug nanoparticles were dispersed within 4-[mu]m water droplets when administered via nebulization or as micron-sized flocs using a pressurized metered dose inhaler (pMDI). Upon deposition in aqueous media, the aerosolized nanoparticle assemblies dissociated into constituent nanoparticles, raising the available surface area for dissolution and increasing dissolution rates, relative to solid particles. Poorly water soluble drug nanoparticles were prepared using a controlled precipitation (CP) or thin film freezing (TFF) process, in which stable nanoparticles (30-300 nm in diameter) with high potencies (>90 wt% drug) were produced by rapidly nucleating drug solutions in the presence of strongly adsorbing polymers or by freezing, respectively. Amorphous, nanoparticles prepared by CP produced stable aqueous dispersions with high fine particle fractions (FPF) of 77% and total emitted doses (TED) of 1.5 mg/min upon nebulization. CP and TFF also produced anisotropic particles (aspect ratios >5), which formed stable suspensions in a hydrofluoroalkane propellant. Inefficient packing of anisotropic particles formed loose, open flocs that stacked upon each other to prevent settling. Upon pMDI actuation, atomized propellant droplets shear apart and template portions of the floc to yield porous particles with high FPFs (49-64%) and TEDs (2.4 mg/actuation). The controlled assembly of gold nanoparticles into clusters is also of great interest for biomedical imaging and therapy because clusters exhibit improved near infrared absorbance (where blood and tissue are most transparent), relative to single spherical particles, and can biodegrade into clearable particles. Gold nanoparticles (5 nm) were assembled into clusters between 30 to 100 nm in diameter with high gold loadings, resulting in strong NIR absorbance. The assembly was kinetically controlled with weakly adsorbing polymers by manipulating electrostatic, van der Waals, steric, and depletion forces. Furthermore, clusters assembled with a biodegradable polymer deaggregated back into primary particles in physiological media and within cells. This kinetic assembly platform is applicable to a wide variety of fields that require high metal loadings and small particle sizes. / text

Controlled drug delivery devices provide numerous advantages such as reduced side effects, higher therapeutic efficiency and improved patient compliance. Biodegradable polymer has become the most important material for controlled drug delivery device because of the excellent biocompatibility and tunable physicochemical properties. Biodegradable polymeric drug delivery devices are usually processed into various types of micro-particles due to the ease of fabrication and administration. However, controlling the drug release kinetics of these microparticles is still a challenge. One important reason is that drug release kinetics is significantly influenced by the microstructure of drug delivery devices, which is difficult to control. 　Microfluidic method is a group of technologies involved in the manipulation of fluids using channels in the scale of micrometers. Microfluidic method is particularly useful in controlling the structure of micro-droplets and generating homogeneous droplets. Therefore, microfluidics suggests great potential in controlling microstructures of drug delivery devices and drug release kinetics. 　In this study, biodegradable polymer based controlled drug delivery devices were fabricated using microfluidic method. Various types of microstructures were developed such as microspheres, core-shell microspheres, hollow microspheres and hydrogel microspheres. The results showed that microstructures were well controlled by fluid flow rates and geometries of capillary microfluidic devices. Both hydrophobic and hydrophilic drugs could be delivered by choosing drug delivery devices with suitable microstructures. 　Drug release kinetics of biodegradable polymeric microspheres has been studies a lot, yet complete understanding is still to be achieved. The diameter is an important factor which contributes to the drug release kinetics. However, the influence of diameter has not been systemically studied because monodisperse microspheres are difficult to obtain. Using microfluidic method, monodisperse PLGA microspheres with different diameters were fabricated to study the influence of diameter on drug release kinetics. It was found that diameter only influence the duration of the first phase (lag phase) in drug release process and smaller microspheres exhibited shorter lag phase. The relatively faster expansion of smaller microspheres was found to be responsible for the size effect by monitoring physicochemical changes during drug release. 　Rifampicin, a broad-spectrum antibiotic, was encapsulated by PLGA microspheres and PLGA-alginate core-shell microspheres. The long-term antimicrobial effects of drug loaded microspheres were investigated by drug release test and antimicrobial test against Staphylococcus aureus. The results showed that drug delivery devices could provide antimicrobial effect for more than one month. These drug delivery devices show potential in applications of controlled drug delivery and long-term antimicrobial therapy. 　In conclusion, drug delivery devices with different microstructures were fabricated using microfluidic method. The diameter of PLGA microspheres only influence the first phase of drug release profile (lag phase) and smaller microspheres exhibited shorter lag phase. The size effect is due to the relatively faster expansion rate of smaller microspheres. Rifampicin loaded PLGA microspheres and PLGA-alginate core-shell microspheres could provide sustained release of rifampicin for more than one month. The released rifampicin was able to inhibit the growth of Staphylococcus aureus. The controlled drug delivery devices presented showed great potential in long-term antimicrobial applications. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Polycationic nanoparticles were synthesized with an activators regenerated by electron transfer for atom transfer radical polymerization-based (ARGET ATRP-based) emulsion in water method and investigated for their utility as biomaterials for drug delivery. The polycationic nanoparticles were composed of 2-(diethylamino)ethyl methacrylate (DEAEMA) for pH-responsiveness, poly(ethylene glycol) methyl ether methacrylate (PEGMA) for improved biocompatibility, tert-butyl methacrylate (tBMA) to impart hydrophobicity, and a tetraethylene glycol dimethacrylate (TEGDMA) cross-linking agent for enhanced colloidal stability. Dynamic light scattering demonstrated pH-responsive swelling, and cell-based assays demonstrated pH-dependent membrane disruption. The polycationic nanoparticles demonstrated low toxicity to cells. The polycationic nanoparticles were evaluated for use as drug delivery biomaterials by investigating the interactions with the drug and cells. Delivery remains a major challenge for translating small interfering RNA (siRNA) to the clinic, and overcoming the delivery challenge requires effective siRNA delivery vehicles. The polycationic nanoparticles demonstrated efficient siRNA loading. Evidence of siRNA-induced knockdown in cells was observed following transfection with the polycationic nanoparticle/siRNA complexes. Imaging techniques confirmed enhanced siRNA internalization using the polycationic nanoparticle/siRNA complexes compared to naked siRNA. An array of polycationic nanoparticles synthesized using ARGET ATRP or UV-initiated polymerization methods was characterized to examine the effect of polymerization method on material properties and the connection to molecular structure. An improved understanding of molecular structure, and its connection to polymerization method and material characteristics, may aid the design of advanced materials. The ARGET ATRP polycationic nanoparticles demonstrated increased nanoscale homogeneity compared to the UV-initiated polymerization polycationic nanoparticles; increased nanoscale heterogeneity in the UV-initiated polymerization polycationic nanoparticles was associated with broader transitions. The polycationic nanoparticles promoted cellular uptake of siRNA and induced knockdown, thus demonstrating potential as siRNA delivery vehicles. The ARGET ATRP method provides an alternative route to creating polycationic nanoparticles with improved nanoscale homogeneity. / text

The influence of the size and morphology of carrier particles on drug dispersion performance from passive dry powder inhalers has been extensively studied topic, and a consensus has been reached regarding the adverse effect that larger carrier particle diameters impart to aerosol performance. However, previous studies have generally employed only a few carrier particle size fractions, generally possessing similar surface characteristics. Accordingly, theories developed to explain the influence of the physical characteristics of carrier particles on performance relied heavily on both extrapolation and interpolation. To fill in the gaps from the literature and simultaneously evaluate the influence of carrier particle size and morphology, a comprehensive study was undertaken using 4 lactose grades, each sieved into 13 contiguous sizes, to prepare 52 formulations incorporating a unique lactose grade-size population. The aerosol performance results indicated that large carrier particles possessing extensive surface roughness can improve drug dispersion, in contrast to what has been previously reported. It is proposed that this may be attributed to mechanical detachment forces arising from collisions between the carrier particle and inhaler during actuation. Based on these observations, a novel dry powder inhaler platform was developed, employing carrier particles much larger (> 1 mm) than previously explored in both the scientific and patent literature. Optimization of this technology required the judicious selection of a carrier material, and following an extensive screening process, low-density polystyrene was selected as a model candidate. Given its low mass, diameters in excess of 5-mm could be employed as carriers while still generating high detachment forces. To minimize drug particle aggregation, a novel drug-coating method employing piezo-assisted particle dispersion was developed to compensate for the reduced surface area of the novel carrier particles. In addition, the selection of a suitable inhalation device prototype was instrumental to the overall performance of the technology. In vitro testing of the novel large carrier particles yielded emitted fractions in excess of 85%, and overall drug delivery of up to 69% of the nominal dose. / text

Recent progress in drug discovery has enabled us to target specific intracellular molecules to achieve therapeutic effects. These next generation therapeutics are often biologics which cannot enter cells by mere diffusion. Therefore it is imperative that drug carriers are efficiently internalized by cells before releasing their cargo. Nanoscale polymeric carriers are particularly suitable for such intra-cellular delivery. Although size and surface-charge has been the most studied parameters for nanocarriers, it is now well appreciated that particle shape also plays a critical role in their transport across physiological barriers. Hence there is increasing interest in fabricating shape-specific polymeric nano and microparticles for efficient delivery of drugs and imaging agents. Nanoimprint lithography methods, such as Jet-and-flash imprint lithography (J-FIL), provide versatile top-down processes to fabricate shape-specific, biocompatible nanoscale hydrogels that can deliver therapeutic and diagnostic molecules in response to disease-specific cues. However, the key challenges in top-down fabrication of such nanocarriers are scalable imprinting with biological and biocompatible materials, ease of particle-surface modification using both aqueous and organic chemistry as well as simple yet biocompatible harvesting. Here we report that a biopolymer-based sacrificial release layer in combination with improved nanocarrier-material formulation can address these challenges. The sacrificial layer improves scalability and ease of imprint-surface modification due to its switchable solubility through simple ion exchange between monovalent and divalent cations. This process enables large-scale bio-nanoimprinting and efficient, one-step harvesting of hydrogel nanoparticles in both water- and organic-based imprint solutions. We also show that when shape is decoupled from volume, charge and composition, mammalian cells preferentially internalize disc-shaped nanohydrogels of higher aspect ratios over nanorods. Interestingly, unlike nanospheres, larger-sized hydrogel nanodiscs and nanorods are internalized more efficiently. Uptake kinetics, efficiency and internalization mechanisms are all shape-dependent and cell-type specific. Although macropinocytosis is used by all cells, epithelial cells uniquely internalize nanodiscs using caveolae pathway. On the other hand, endothelial cells use clathrin-mediated uptake along with macropinocytosis for all shapes and show significantly higher uptake efficiency compared to epithelial cells. We also study the effect of shape and surface properties for their tissue uptake and penetration using spheroids as a 3D tumor model and show that hydrophobic particles show no difference in penetration inside such models even after 125 fold reduction in volume. These results provide a fundamental understanding of how cell and tissue behavior is influenced by nanoscale shape and surface properties and are critical for designing improved nanocarriers and predicting nanomaterial toxicity. / text

Cardiovascular disease (CVD) and particularly atherosclerosis is a leading cause of morbidity in the developed world. Atherosclerosis and the rupture of vulnerable atherosclerotic plaque cause 70% of deaths from CVD. The progression of atherosclerosis has been identified as a pathological inflammatory process. Targeting atherosclerotic drug therapies to inflammatory markers has emerged as an important and growing research area. The adhesion molecule CD44 has been implicated in the onset and build-up of atherosclerotic lesions throughout the course of development. The research in this dissertation is aimed at targeting anti-inflammatory therapy to activated vascular endothelium with directed with an anti-CD44 antibody, IM7, conjugated to a non ionic surfactant vesicle (niosome) drug carrier. The IM7 conjugated immunoniosome has been shown to bind to endothelial and synovial lining cells in vitro. The preliminary research is involved with the development of the drug delivery vesicle, and the antibody linkage chemistry, along with an analysis of vesicle characteristics and stability. A novel linking chemistry using polyoxyethylene sorbitan monostearate and cyanuric chloride allows antibodies to be conjugated to vesicle surface polymer groups without prior derivatization. Subsequent research tested the resulting 'immunoniosome's' ability to bind to target antigens with selectivity and specificity. Bovine aortic endothelial cells activated with cytokines provide a model of inflammation. Analysis of binding was done through fluorescent and scanning electron microscopy. In vivo uptake of vesicles at sites of inflammation is size dependent. In order to overcome this barrier to uptake, niosome suspensions were thermally extruded to create uniform 200 nm vesicles. Further analysis of the efficacy of the system looked at live cell uptake of the immunoniosomes measured by confocal and transmission electron microscopy. Preparation for in vivo murine studies required that the antibody component was modified to counteract the immune response. Finally, the conjugation of antibody fragments to niosomes and the binding and uptake of the vesicles in a live endothelial cell model is evaluated. A viable drug delivery particle showing binding and cellular uptake capabilities in inflammatory cells was produced by this research using a novel surfactant-antibody linker.