The local release of complexed siRNA from biomaterials enables targeted therapy of specific cells and tissues. This thesis focused on gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA) as a drug delivery system for siRNA. The siRNA-loaded cGM were aggregated with SaOS-2 cells or human mesenchymal stem cells (hMSC) to microtissues and stimulated with osteogenic supplements. Cell survival and tissue formation in microtissues could be improved by incorporating cGM in spheroid cultures. We observed hydroxyapatite deposition in the particles in dependence of medium and cell type. Osteogenic stimulation with BMP-2 and simultaneous silencing of BMP-2 antagonist chordin accelerated matrix mineralization of the microtissues. Higher cross-linking degree of cGM positively influenced chordin silencing and alkaline phosphatase (ALP) activity as a marker for osteogenic differentiation. These higher cross-linked cGM mineralized in an osteogenic medium within 8–9 days, in presence and absence of cells. The effects of pre-differentiated and chordin-silenced microtissues were investigated by simulation of in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). Increased ALP activity and osteoprotegerin (OPG) secretion were observed after 14 days compared to co-cultures with siRNA-free controls. These results indicate that the pre-differentiated and silenced microtissues can induce osteogenic differentiation of surrounding unstimulated cells. Using the microtissue approach with siRNA complexed with tyrosine-modified low molecular weight polyethyleneimine (P10Y/P5Y) as transfection reagent was not successful. The results of this thesis indicate that the pre-differentiation of microtissues with BMP-2 in combination with chordin silencing stimulates and enhances osteogenic differentiation of other stem cells. As a combination of biomaterial, RNAi, and autologous cells, microtissues could be a promising approach to regenerating bone defects.:Chapter I: Controlled release of siRNA for bone tissue engineering Chapter II: Microtissues from mesenchymal stem cells and siRNA-loaded cross-linked gelatin microparticles for bone regeneration Chapter III: Mineralizing gelatin microparticles as cell carrier and drug delivery system for siRNA for bone tissue engineering Chapter IV: Tyrosine-modified polyethylenimines for siRNA transfection in microtissues Chapter IV: Final discussion

A thesis submitted to the School of Graduate Studies in partial fulfilment of the requirements for the degree of Master of Science in the graduate academic unit of Biology / Microparticles enter aquatic environments through many sources, including wastewater treatment plants (WWTPs), but their uptake by aquatic organisms is poorly understood. Freshwater bivalves accumulate multiple contaminants, making them potential bioindicators for MP pollution. This study aims to understand the abundance and characteristics of microparticles that accumulate in wild bivalves. Samples were collected from 5 locations along the Grand River (Ontario, Canada) in 2021-2022, including 3 municipal WWTPs where both an upstream and downstream site were sampled. At each site, fingernail clams (Sphaeriidae, n=5 composite samples), flutedshell mussels (Lasmigona costata, n=10), and surface water (n=3) were sampled. Within the mussels, the gill, digestive gland, and hemolymph tissues were targeted and compared. Microparticles were isolated and quantified via stereomicroscopy but have not yet been confirmed as plastic; as such, they will be referred to herein as microparticles. Fibers were the dominant morphology and clear, blue, and black were the most common colours, but there were some differences among sites and sample types. Most microparticles were between 80 μm and 1 mm in length. Fingernail clams contained the highest microparticle counts per mass of tissue at 35.5 ± 29.4 microparticles/g, mussel tissues ranged from 4.3 ± 4.2 microparticles/mL to 6.5 ± 8.1 microparticles/g, and water samples contained the lowest counts at 0.0055 ± 0.0028 microparticles/mL. Elevated microparticle counts at downstream sites were only seen in mussel gills and not other bivalve tissues. Surface water samples did not show elevated counts downstream of the WWTPs and microparticle exposures were similar across sites. This study provides baseline data for future monitoring and informs toxicity studies to fully assess the risk of microparticles to vulnerable freshwater bivalves and other aquatic organisms. It also suggests microparticles in freshwater bivalves are coming from sources in addition to WWTPs and are ubiquitous in the Grand River. / Thesis / Master of Science (MSc) / Microplastics are found in nearly every environment, especially freshwater ecosystems. These plastics come from a variety of sources, and this study focuses on assessing the characteristics of microparticles in freshwater clams and mussels (bivalves) that have been exposed to municipal wastewaters. Bivalves and water samples were collected from 5 locations along the Grand River (Ontario, Canada) in 2021-2022, and microparticles were extracted and analyzed from each sample. Fibers were the most abundant type of microparticle, with colours consisting mostly of clear, blue, and black. Clams had the highest number of microparticles per mass of tissue collected and the lowest counts were found in water samples. Higher microparticle counts were only seen in one (mussel gill) of the four tissues from bivalves collected downstream of wastewater outfalls. This study provides baseline data on microparticle characteristics in freshwater bivalves and will guide future studies on the toxicity of microparticles to these animals.

No description available.

No description available.

Magnetic particles with active functional groups offer numerous advantages for use in μ-TAS (Micro Total Analytical Systems). The functional site allows chemical binding of the particle with the target species in the fluid sample. Selection of the functional group establishes the target molecule and vice versa under assumptions of highly specific biding. The particles hence act as mobile reaction substrates with high surface to volume ratios owing to their small size. The concept of action at a distance allows their use as agents for separation in microchannels based on relatively simple design. It is possible to manipulate magnetic particles and bound target species using an externally applied magnetic field. Hence, the particles can be effectively separated from the flow of a carrier fluid. Magnetic fields create dipolar interactions causing the particles to form interesting structures and aggregates. Depending upon the applied field, the microstructure evolution of the aggregate is interesting in its own right, e.g. related to improvements in material properties and bottom-up self assembly. The shape of the aggregates can be determined a priori if the interaction between the particles is well characterized. The dominant competing forces that influence magnetic particle dynamics in a flow are magnetic and viscous. There are a number of physical parameters such as viscosity, magnetic susceptibility, fluid velocity, etc. which are varied to study their individual effects. Initially dilute suspensions are studied experimentally and numerically using a particle based dynamics approach. Once established, a force model for particle interaction is investigated for concentrated suspensions. A Lagrangian particle tracking algorithm that returns positions of the particles is used for this work that focuses on studying the dynamics of these particles. A mathematical model is proposed and investigated for functionalization between magnetic and non-magnetic particles. Having characterized the collection of magnetic particles, the effect of relative concentrations is investigated on the collection of the non-magnetic species. / Ph. D.

As the world population ages, hospital discharges of geriatric patients to nursing homes have increased. Patients with peripherally inserted central catheters (PICCs) are routinely discharged with the catheters in place. PICCs, only capable of being tracked through x-ray imaging, will routinely experience complications due to thrombosis or accidental dislodgement from poor at-home care. Routinely, elderly patients will be forced to revisit the hospital to have the catheter replaced using x-ray imaging, exposing them to hospital borne illness. Catheters with the capability to be tracked without the need of x-ray imaging would greatly benefit the ill and elderly, providing decreased stress to the patients and increase nursing home capabilities. This project seeks to develop the field of real-time ultrasound tracking of polymeric medical devices, through fabrication of ultrasound responsive polymer-glass composites. Optimal composition will be researched through three complimentary approaches. The first approach seeks to develop a polyurethane-glass microparticle composite to understand the relationship between microparticle loading and ultrasound imaging. In the second approach, manufacturing and end-use complications will be simulated to evaluate the effects on mechanical and ultrasonic properties. Furthermore, impacts from in-vitro long term catheterization to the sample mechanical and ultrasound morphologies would be analyzed. In the third approach, optimization from the previous approaches would assist in the replacement of medical grade polyurethane with medical grade thermoset silicone in hopes to prove the ability for the research to be transferable to other medical polymeric devices. The stated approaches will be useful for setting a path towards the development of ultrasonic imaging as the standard for medical device tracking. / Doctor of Philosophy / As the world population ages, hospital discharges of geriatric patients to nursing homes have increased. Patients with peripherally inserted central catheters (PICCs) are routinely discharged with the catheters in place. PICCs, only capable of being tracked through x-ray imaging, will routinely experience complications due to poor at-home care. Routinely, elderly patients will be forced to revisit the hospital to have the catheter replaced using x-ray imaging, exposing them to hospital borne illness. Catheters with the capability to be tracked without the need of x-ray imaging would greatly benefit the ill and elderly, providing decreased stress to the patients and increase nursing home capabilities. We hope to develop the field of real-time ultrasound tracking of plastic medical devices, through production of ultrasound activated plastic devices.

Microrobotics is an emerging discipline with the potential to radically affect fields ranging from medicine to environmental stewardship. Already, there have been remarkable breakthroughs; small scale robots have been made that can selectively traverse the gastrointestinal tract, and others have been built that can fly in a manner inspired from bees. However, there are still significant challenges in microrobotics, and it remains difficult to engineer reliable power sources, actuators, and sensors to create robust, modular designs at the microscale. The miniaturization of the robotic system makes design and efficiency of these components particularly difficult. However, biological systems demonstrate the key features of robotics " sensing, actuation, processing" and are remarkably complex at the microscale. As such, many researchers have turned to biology for inspiration and living robotic components. In our laboratory we have engineered an Escherichia coli (E. coli) capable of producing surface display proteins to either anchor the cells, bind to functionalized nanoparticles, or capture small molecules from the environment, all complex actuation features. Additionally, we have created a processing unit that can create signals based on biological components, yet is non-living. This thesis focuses on the characterization of the surface display E. Coli system and the creation of programmable microparticle scaffolds that may be controlled by biological circuitry. In particular, by leveraging the strong interaction between biotin and streptavidin, I have created programmable microparticle scaffolds capable of attenuating the intensity of a fluorescent response in response to perturbations in the local environmental conditions. We believe this is an excellent enabling technology to facilitate the creation of complex behaviors at the microscale and can be used as a processing unit for simple decision making on microrobots. We foresee this technology impacting disciplines from medical microrobotics to environmental sensing and remediation. / Master of Science / Robots have integrated into industries ranging from car manufacturing to in-hospital transportation. However, recently there has become a new desire for robotics at a smaller scale, for use in fields ranging from medicine to agriculture. For example, how awesome would it be if we had small robots traveling through our bloodstream giving therapeutic drugs as needed or selectively killing tumor cells? This is the dream and goal of many research labs currently. However, when we try to design these tiny robots, we find that we are unable to use many of the normal components that are seen when looking at conventional electronics, AAA batteries as power sources for example. To build upon the example, how would a tiny robot in the body power itself or know when it has reached a cancerous tumor? We propose that the problem of decreased space can be solved by using biological components, like bacteria cells which already live at a microscopic scale, to power these robots and help them sense their surroundings. The work discussed in this thesis involves the design of biological sensors and processing units. We have proven that by engineering the DNA of bacteria cells, using the tools of synthetic biology, we are able to use the outside of the cell (cell’s surface) to sense components in the environment. We hope that the findings discussed in this thesis will serve as the ground work for integrating living cells and robotics for future applications ranging from medicine to environmental remediation.

Yes / Platelet microparticle (PMP)-induced angiogenesis plays a key role in tumour metastasis and has been proposed to contribute towards cardiovascular disease by enhancing atherosclerotic plaque vulnerability. However, the mechanisms underlying PMP induced angiogenesis are ill defined. Recent reports demonstrate that PMPs deliver micro-RNAs (miRNAs) to recipient cells, controlling gene expression. We therefore evaluated whether miRNA transfer was a key regulator of PMP-induced angiogenesis. Co-culturing PMPs with human umbilical vein endothelial cells (HUVEC) on extracellular matrix gel induced robust capillary like structure formation. PMP treatment altered the release of angiogenesis modulators from HUVEC, including significantly reducing production of anti-angiogenic thrombospondin-1 (THBS-1). Both functional responses were abrogated by treating PMPs with RNase, suggesting the transfer of PMP-derived RNA was a critical event. PMPs were an abundant source of miRNA Let-7a, which was transferred to HUVEC following co-incubation. Using luciferase reporter assays we have shown that Let-7a directly targets the 3’UTR of the THBS-1 mRNA. HUVEC transfection with a Let-7a anti-sense oligonucleotide reduced the ability of PMPs to inhibit THBS-1 release, and significantly decreased PMP induced in vitro angiogenesis. Antibody neutralisation of THBS-1 reversed the anti-angiogenic effect of let-7a inhibition in PMP treated HUVEC, highlighting Let-7a dependent translational repression of THBS-1 drives angiogenesis. Importantly, plasmid overexpression of Let-7a in HUVEC alone induced robust tubule formation on extracellular matrix gel. These data reveal a new role for Let-7a in promoting angiogenesis and show for the first time PMPs induced angiogenic responses occur through miRNA regulation of HUVEC.

Embryonic stem cells (ESCs) are a unique cell population that can differentiate into all three embryonic germ layers (endoderm, mesoderm, and ectoderm), rendering them an invaluable cell source for studying the molecular mechanisms of embryogenesis. Signaling molecules that direct tissue patterning during embryonic development are secreted by ESC aggregates, known as embryoid bodies (EBs). As many of these signaling proteins interact with the extracellular matrix (ECM), manipulation of the ESC extracellular environment provides a means to direct differentiation. ECM components, such as glycosaminoglycans (GAGs), play crucial roles in cell signaling and regulation of morphogen gradients during early development through binding and concentration of secreted growth factors. Thus, engineered biomaterials fabricated from highly sulfated GAGs, such as heparin, provide matrices for manipulation and efficient capture of ESC morphogens via reversible electrostatic and affinity interactions. Ultimately, biomaterials designed to efficiently capture and retain morphogenic factors offer an attractive platform to enhance the differentiation of ESCs toward defined cell types. The overall objective of this work was to examine the ability of microparticles synthesized from both synthetic and naturally-derived materials to enhance the local presentation of morphogens to direct ESC differentiation. The overall hypothesis was that microparticles that mimic the ECM can modulate ESC differentiation through sequestration of endogenous morphogens present within the EB microenvironment.

As parasitoses causadas por helmintos constituem um grave problema sanitário, tanto para os seres humanos quanto para os animais, além de gerar grandes prejuízos econômicos. O sulfóxido de albendazol é um fármaco anti-helmíntico de amplo espectro, largamente utilizado na medicina veterinária, veiculado pelas vias oral e parenteral, mediante a utilização de formas farmacêuticas convencionais. Apresenta biodisponibilidade baixa e irregular em função de sua pouca solubilidade nos fluidos biológicos. Para a manutenção da concentração plasmática e completa eliminação dos parasitos, são necessárias administrações reiteradas, ocasionando transtornos decorrentes do manejo freqüente dos animais e do aumento do custo da terapia. O presente trabalho teve como objetivo desenvolver e caracterizar um sistema microparticulado para liberação sustentada de sulfóxido de albendazol, de modo que este pudesse permanecer no organismo dos animais pelo tempo suficiente para a completa eliminação dos parasitos após uma única administração. Os sistemas foram obtidos a utilizando as técnicas de spray-drying e emulsificação / evaporação de solvente, tendo sido utilizados os polímeros Eudragit RS 30 D® e Eudragit RS PO®, respectivamente. As micropartículas obtidas foram caracterizadas com relação ao tamanho, à morfologia e à eficiência de encapsulação. Através da técnica de emulsificação / evaporação de solvente, foram obtidas partículas com diâmetro médio inferior a 300nm, estreita faixa de distribuição de tamanho e eficiência de encapsulação de aproximadamente 60%. Os resultados do estudo in vitro do perfil de liberação do fármaco a partir das micropartículas obtidas mostraram que, apesar de o sistema desenvolvido não ter sido capaz de sustentar a liberação do fármaco, o mesmo promoveu um aumento significativo da solubilidade do sulfóxido de albendazol em pH 7,4, fato este que pode contribuir para o aumento da biodisponibilidade do mesmo após administração parenteral. / The helminthosis are a serious sanitary problem, as for the men than for the animals, besides the great economic lacks. Albendazole sulphoxide is an antihelminthic drug with broad spectrum of action, widely used at veterinarian medicine, throw oral and parentereal vies, in conventional pharmaceutical dosages. It has low and irregular bioavailability due its low solubility in the biological fluids. For the maintenance of the plasmatic concentration and complete elimination of the parasites, it is required several administrations, creating many troubles due the frequent handling of the animals and increase in the costs of the therapy. The present work had as objective to develop and characterize microparticles for sustained release of albendazole sulphoxide, in order that the drug could be for a longer time in the animals organisms and the parasites could be eliminated after just one administration. The referred microparticles were obtained from the spray-drying and emulsification / evaporation of solvent techniques, using the polymers Eudragit RS 30 D® and Eudragit RS PO®, respectively. The obtained systems were characterized considering size, morphology and encapsulation efficiency. Using the emulsification / evaporation of solvent technique, it was prepared microparticles with medium diameter under 300nm, narrow range of size distribution and encapsulation efficiency of about 60%. The results of the in vitro release profile study of the drug from the prepared microparticles showed that besides the developed system was not be able to sustain the drug delivery, it was able to improve significantly the solubility of albendazole sulphoxide at pH 7.4, what can be useful to improve its parenteral bioavailability.