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<p>Coated calcium-alginate beads are the basis of many encapsulation methods used in pursuit of cell-based enzyme and hormone replacement therapies. The standard alginate - poly-L-lysine - alginate (APA) capsules consist of a calcium-alginate hydrogel core containing cells designed to express a therapeutic product, coated with permeability controlling poly-L-lysine (PLL, a polycation) followed by an exterior layer of polyanionic alginate. Although this approach is promising, the required long-term survival of the implanted cells has remained largely elusive as the current APA capsules suffer from several biocompatibility and mechanical strength issues, one of which is the weakening of ionic crosslinks over time, exposing the encapsulated cells to the host.</p> <p>This thesis aims to replace the exterior layer of alginate with a Temporarily Reactive Polyelectrolyte (TPR) to reinforce AP capsules by forming covalently crosslinked shells. TRPs are polyanions that possess reactive electrophilic groups capable of forming permanent covalent crosslinks with the underlying polyamine (such as PLL), and subsequently hydrolyze, increasing the net negative charge of the polyanion. TRPs are thought to improve the biocompatibility and strength of the microcapsules by forming stable inert amide bonds, as well as increasing the net negative charge of the capsule through the liberation of carboxylates. This thesis will focus primarily on two TRPs: 50% hydrolyzed poly(methyl vinyl ether-<em>alt</em>-maleic anhydride), PMM₅₀ , and poly(methacrylic acid-co-2-vinyl-4,4-dimethylazlactone) with a 50:50 co-monomer ratio, PMV₅₀ . Their synthesis, rates of hydrolysis and capsule formation around encapsulated C2C12 cells for <em>in-vitro</em> and<em> in-vivo</em> studies will be described. Additionally the synthesis and rates of hydrolysis of other 2-vinyl-4,4-dimethylazlactone (VDMA)-copolymers are presented as potential candidates for future TRPs.</p> / Doctor of Philosophy (PhD)

Grafenoxid (GO) har potential att användas i läkemedelsleveransapplikationer. Dess stora specifika yta gör det intressant som en effektiv bärare och skyddare av olika aktiva substanser för genterapi, såsom DNA och mRNA. Denna studie har fokuserat på att undersöka förhållandena för att ladda negativt laddat mRNA på GO. Kitosan (CS) och linjär polyetylenimin (PEI) har preadsorberats på GO för att underlätta mRNA-adsorption. Studien undersökte vid vilka förhållanden zeta-potentialen av GO/polyelektrolyt för det negativt laddade GO blir positivt. Dessa komplexerades sedan med mRNA vid olika N/P-förhållanden. Dessutom bedömde studien mRNA-frisättningskapaciteten genom att reducera pH.  GO/CS-komplexet vid förhållandet 1:2 visade positiv zeta-potential med N/P-förhållandena som sträcker sig från 1:1 till 10:1 visade att all mRNA och polyA har adsorberat till komplexet. N/P-förhållandet 10:1 var den enda som uppnådde en neutral zeta-potential, vilket tyder på tillräckligt mRNA för mättnad. Genom att öka koncentrationen av CS, kunde zeta-potentialen skifta till positivt vilket potentiellt förbättrar transfektionseffektiviteten. Visade en förbättring i signalen av det fria mRNA ökade när GO/CS/mRNA-komplexet utsattes för ett mer surt pH. Detta tyder på en potentiell frisättning när vektorn transfekteras in i cellen, eftersom den transporteras till lysosomerna som kännetecknas av sin sura miljö. GO/PEI-komplex visade endast negativ zeta-potential vid GO:PEI-förhållanden som når upp till 1:10, och därmed kommer det negativt laddade mRNA inte att adsorbera på dessa GO/PEI-komplex.  Resultaten tyder på en lovande utgångspunkt för pre-formuleringen av GO/CS-komplexet för vidare forskning. Detta arbete ger ett bidrag för framtida studier inom detta område. / Graphene oxide (GO) has a potential to be used in drug delivery applications. The large surface-to-mass ratio makes it interesting as efficient carrier and protector of various substances aimed for therapy, including DNA and mRNA. This study has focused on determining the ideal conditions for loading negatively charged mRNA onto GO using chitosan (CS) and linear polyethyleneimine (PEI) to facilitate mRNA adhesion. This was achieved by examining at what ratios of GO/polyelectrolyte the zeta potential of the negatively charged GO becomes positive, which were then subjected to mRNA complexation at different N/P (nitrogen/phosphate) ratios. Moreover, the study assessed the mRNA release capability by altering the pH.  The GO/CS complex at ratio 1:2 showed positive zeta potential with the N/P ratios ranging from 1:1 to 10:1 presented 100% loading efficiency of the added nucleic acids. With the N/P ratio 10:1 standing out as it achieved a neutral zeta potential, suggesting enough mRNA for saturation. By increasing the concentration of CS, the zeta potential could shift to positive potentially enhancing transfection efficiency. During the release assessment, the GO/CS/mRNA complex displayed increased amount of unbound mRNA when subjected to a more acidic pH. This suggests potential release when transfected into the cell, as the vector is transported to the lysosomes characterized by their acidic environment. GO/PEI complexes demonstrated only negative zeta potential at GO:PEI ratios reaching to 1:10, and thus the negatively mRNA will not adsorb on these GO/PEI complexes.  The findings suggest a promising starting point for the pre-formulation of the GO/CS complex for further research. This work provides a solid foundation for future studies in this area.

Touch (or tactile) sensors are gaining renewed interest as the level of sophistication in the application of minimally invasive surgery and humanoid robots increases. The spatial resolution of current large-area tactile sensors (greater than 1 cm2) lag human fingers by over an order of magnitude. Using metal and semiconducting nanoparticles, a ~100 nm thick, large area thin-film device working on the principles of electron tunneling is self-assembled, such that the change in current density through the film and the electroluminescence light intensity are linearly proportional to the local stress. By pressing a United States 1 cent coin (and also a copper grid) on the device a well resolved stress image by focusing the electroluminescence light directly on CCD is obtained. Both the lateral and height resolution of texture are comparable to human finger at similar stress levels of ~10 KPa. The fabrication of the film is based on self-assembly of polyelectrolytes, and metal and semiconducting nanoparticles in a layered architecture. The polyelectrolyte layer functions as the dielectric tunneling barrier and the nanoparticles function as the base for tunneling electrons. The assembly of the device can be simplified by incorporating the functionality of the polyelectrolyte and the nanoparticles in a single composite medium. A non-micellar mineralization process for the synthesis of multifunctional nanocomposite materials is also reported as a possible building block for the assembly of tactile sensor. The non-micellar method results in the synthesis of monodisperse semi-conducting nanoparticles templated on polymer chains dissolved in solution at high yield. The monodispersity is achieved due to the beaded necklace morphology of the polyelectrolyte chains in solution where the beads are nanometer-scale nodules in the polymer chain and the nanoparticles are confined to the beads. The resultant structure is a nanoparticle studded necklace where the particles are imbedded in the beads. Multiple cycles of the synthesis on the polymer template yield nanoparticles of identical size, resulting in a nanocomposite with high particle fraction. The resultant nanocomposite has beaded-fibrilar morphology with imbedded nanoparticles, and can be solution cast to make electroluminescent thin film devices. The concept is further modified for synthesis of metal nanoparticles on polyelectrolyte templates with isolated beaded morphology. / Ph. D.

Fiber-optic Fabry-Perot interferometric (FFPI) sensors have been widely used due to their high sensitivity, ease of fabrication, miniature size, and capability for multiplexing. However, direct measurement of self-assembled thin films, receptor immobilization process or biological reaction is limited in the FFPI technique due to the difficulty of forming Fabry-Perot cavities by the thin film itself. Novel methods are needed to provide an accurate and reliable measurement for monitoring the thin-film growth in the nanometer range and under various conditions. In this work, two types of fiber-optic multicavity Fabry-Perot interferometric (MFPI) sensors with built-in temperature compensation were designed and fabricated for thin-film measurement, with applications in chemical and biological sensing. Both the tubing-based MFPI sensor and microgap MFPI sensor provide simple, yet high performance solutions for thin-film sensing. The temperature dependence of the sensing cavity is compensated by extracting the temperature information from a second multiplexed cavity. This provides the opportunity to examine the thin-film characteristics under different environment temperatures. To demonstrate the potential of this structure for practical applications, immunosensors were fabricated and tested using these structures. Self-assembled polyelectrolytes served as a precursor film for immobilization of antibodies to ensure they retain their biological activity. This not only provides a convenient method for protein immobilization but also presents the possibility of increasing the binding capacity and sensitivity by incorporating multilayers of antibodies into polyelectrolyte layers. The steady-state measurement demonstrated the surface concentration and binding ratio of the immunoreaction. Analysis of the kinetic binding profile provided a fast and effective way to measure antigen concentration. Monitoring the immunoreaction between commercially available immunoglobulin G (IgG) and anti-IgG demonstrated the feasibility of using the MFPI sensing system for immunosensing applications. / Ph. D.

We report the assembly of three-dimensional (3D) liver sinusoidal mimics comprised of primary rat hepatocytes, human or rat liver sinusoidal endothelial cells denoted as hLSECs and rLSECs respectively, and an intermediate chitosan-hyaluronic acid (HA) polyelectrolyte multilayer (PEM). The height of the PEMs ranged from 30-55nm and exhibited a shear modulus of ~ 100kPa. Primary rat hepatocytes coated with 5 and 15 PE layers exhibited stable urea and albumin production over a seven day period and these values were either comparable or superior to that in a collagen sandwich (CS). Hepatocyte-PEM-hLSEC liver mimics exhibited stable urea production and increasing albumin secretion over the culture period in comparison to hepatocyte-LSEC samples. In the 3D liver mimics, hLSEC phenotype was maintained and verified by the uptake of acetylated low-density lipoprotein (AcLDL). A sixteen-fold increase in CYP1A1/2 activity was observed for hepatocyte-PEM-10,000 hLSEC samples, thereby, suggesting that interactions between hepatocytes and hLSECs play a key role in enhancing hepatic phenotypes in in vitro cultures. As the first step towards elucidating key signaling pathways involved in cell-cell communications, global genome-wide transcriptional profiles of primary hepatocytes cultured in CS and hepatocyte monolayers (HMs) were performed over an eight-day period using DNA microarray measurements and Gene Set Enrichment Analysis (GSEA) in order to derive biologically meaningful information at the level of gene sets. The gene expression in CS cultures steadily diverged from that in HMs. Gene sets up-regulated in CS are those linked to liver metabolic and synthetic functions, such as lipid, fatty acid, alcohol and carbohydrate metabolism, urea production, and synthesis of bile acids. Monooxygenases such as CYP enzymes were significantly up-regulated starting on day 3 in CS cultures. These results serve as a baseline for further investigation into the systems biology of engineered liver tissues. 3D hepatic constructs were also assembled with primary rat hepatocytes and rLSECs, and a chitosan-HA PEM. In these hepatic models, the phenotype of hepatocytes and rLSECs were maintained. rLSEC phenotype was verified over a twelve-day period through immunostaining with the sinusoidal endothelial-1 (SE-1) antibody. In contrast, rLSECs cultured as monolayers lost their phenotype within 3 days. A two-fold increase in albumin production was observed only in the 3D liver models. rLSEC-PEM-hepatocyte cultures exhibited three- to six-fold increased CYP1A1/2 and CYP3A enzymatic activity. Well-defined bile canaliculi were observed in only 3D hepatic constructs. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte constructs can be used as liver models for future studies. / Ph. D.

The formation of layered nanoparticle films on the surface of wood fibers is reported in this study. The layer-by-layer (LbL) assembly technique was comprehensively investigated as a non-covalent surface modification method for lignocellulosic fiber. Nanocomposite-based lignocellulosic fibers were successfully fabricated by sequential adsorption of oppositely charged poly(diallydimethylammonium) chloride (PDDA) and clay nanoparticles in a number of repeated deposition cycles. Nanocomposite fibers displayed layered structure as indicated by the electrokinetic potential studies and scanning electron microscopy (SEM) analysis. Layer-by-layer films of PDDA and clay impacted the thermal stability of wood fibers. Average degradation temperature at 5 and 10% weight loss for modified fibers with 4 bi-layers increased by up to ~24 and ~15°C, respectively. Significant char residue formed for the LbL modified fibers after heating to 800°C, indicating that the clay-based coating may serve as a barrier, creating an insulating layer to prevent further decomposition of the material. Layer-by-layer film formation on wood fibers was investigated as a function of parameters related to fiber composition and solution conditions (ie. presence of lignin, salt concentration and pH). Elemental analysis of modified fibers revealed that PDDA adsorption to the fibers was reduced for all solution conditions for the samples with the highest content of lignin. Upon extracting the non-covalently attached lignin, the samples showed the greatest amount of PDDA adsorption, reaching to 1.5% of total mass, under neutral solution conditions without the presence of added electrolyte. Furthermore, the influence of both the amount of PDDA adsorbed onto the fiber surface and electrokinetic potential of modified fibers on subsequent multilayer formation was quantified. Under select fiber treatments, great amount of PDDA/clay (up to ~75% total mass for only 4 bi-layers) was adsorbed onto wood fibers through the LbL process, giving these high surface area fibers nanocomposite coatings. LbL modified fibers were melt compounded with isotactic polypropylene (PP) and compression molded into test specimens. The effect of LbL modification as a function of the number of bi-layers on composite performance was tested using the tensile, flexural, dynamic mechanical and thermal properties of fiber reinforced thermoplastic composites. LbL modified fiber composites had similar modulus values but significantly lower strength values than those of unmodified fiber composites. However, composites composed of LbL modified fibers displayed increased elongation at break, increasing by more than 50%, to those of unmodified samples. DSC results indicated that crystallization behavior of PP is promoted in the presence of wood fibers. Both unmodified and LbL modified fibers are able to acts as nucleating agents, which cause an increase of the crystallinity of PP. Moreover, results from tensile and flexural strength, dynamic mechanical analysis and water absorption tests revealed that the material (PDDA or clay) at the terminal (outer) layer of LbL modified fiber influences the performance of the composites. These findings demonstrate control over the deposition of nanoparticles onto lignocellulosic fibers influencing terminal surface chemistry of the fiber. Further investigation into using renewable fibers as carriers of nanoparticle films to improve fiber durability, compounding with thermoplastics that have higher melt processing temperatures, and tailoring terminal surface chemistry to enhance adhesion is justified by this research. / Ph. D.