Browsing by Subject "hydrogel"
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Item Development of a "Self-Cleaning" Encapsulation Technology for Implantable Glucose Monitoring(2011-02-22) Gant, Rebecca M.The increasing prevalence of diabetes and the severity of long-term complications have emphasized the need for continuous glucose monitoring. Optically-based methods are advantageous as they have potential for noninvasive or minimally invasive detection. Fluorescence-based affinity assays, in particular, can be fast, reagentless, and highly specific. Poly(ethylene glycol) (PEG) microspheres have been used to encapsulate such fluorescently labeled molecules in a hydrogel matrix for implantation into the body. The matrix is designed to retain the sensing molecules while simultaneously allowing sufficient analyte diffusion. Sensing assays which depend upon a spatial displacement of molecules, however, experience limited motility and diminished sensor response in a dense matrix. In order to overcome this, a process of hydrogel microporation has been developed to create cavities within the PEG that contain the assay components in solution, providing improved motility for large sensing elements, while limiting leaching and increasing sensor lifetime. For an implanted sensor to be successful in vivo, it should exhibit long-term stability and functionality. Even biocompatible materials that have no toxic effect on surrounding tissues elicit a host response. Over time, a fibrous capsule forms around the implant, slowing diffusion of the target analyte to the sensor and limiting optical signal propagation. To prevent this biofouling, a thermoresponsive nanocomposite hydrogel based on poly(N-isopropylacrylamide) was developed to create a self-cleaning sensor membrane. These hydrogels exist in a swollen state at temperatures below the volume phase transition temperature (VPTT) and become increasingly hydrophobic as the temperature is raised. Upon thermal cycling around the VPTT, these hydrogels exhibit significant cell release in vitro. However, the VPTT of the original formula was around 33-34 degrees C, resulting in a gel that is in a collapsed state, ultimately limiting glucose diffusion at body temperature. The hydrogel was modified by introducing a hydrophilic comonomer, N-vinylpyrrolidone (NVP), to raise the VPTT above body temperature. The new formulation was optimized with regard to diffusion, mechanical strength, and cell releasing capabilities under physiological conditions. Overall, this system is a promising method to translate a glucose-sensitive assay from the cuvette to the clinic for minimally invasive continuous glucose sensing.Item Development of hyaluronic acid – poly(ethylene glycol) hydrogels towards hematopoietic differentiation of mouse embryonic stem cells(2009-08) Erickson, Kathryn Marie; Roy, Krishnendu; Suggs, LauraThe fields of tissue engineering, regenerative medicine, and stem cell engineering are rapidly growing. However, these fields must overcome several obstacles before they can make a significant impact on treating cellular disorders. Two major hurdles that must be addressed are: determining how to control the pluripotency of stem cells and developing systems for high-throughput culture of stem cells. The prospect of using a cell source capable of differentiating into cells of any tissue in the body (embryonic stem cells) has received enormous interest in recent years. The pluripotent attribute of embryonic stem cells seems ideal but developing methods to drive embryonic stem cells to specific lineages, including the hematopoietic lineage, is a complex process dependent on multiple intrinsic and extrinsic factors including chemical, cellular, and environmental signaling. With regards to environmental signaling, the use of three-dimensional culture systems such as scaffolds and hydrogels, have been utilized in an attempt to drive lineage-specific differentiation in a synthetic, biomimetic microenvironment. To determine specific environmental factors responsible for hematopoietic differentiation a systematic biological and engineering process must be implemented. A biodegradable hydrogel composed of the hyaluronic acid, a polysaccharide abundant in the bone marrow microenvironment, and the synthetic polymer, poly(ethylene glycol) was formulated to culture mouse embryonic stem cells (mESCs). Photoencapsulation of mESCs did not significantly decrease cellular viability or proliferation. The FACS data was inconclusive however, from gene expression studies, it was determined that the hydrogel culture system promoted differentiation of mESCs as evidenced by a down-regulation of the gene encoding for stem cell maintenance transcription factor, Oct-3/4. Furthermore, embryoid bodies, necessary for in vitro differentiation were observed in the hydrogel systems. Although an increase in the gene encoding for the cell surface marker, c-kit was up-regulated, the surface marker, sca-1 was not up-regulated. Up-regulation of both c-kit and sca-1 is necessary for the development of hematopoietic progenitor cells. Results indicate that the differentiation of mESCs into the hematopoietic lineage was unsuccessful but differentiation in these hydrogel systems did occur. Future cell marker and gene expression studies are necessary to determine which cell lineage the encapsulated mESCs are differentiating into before the effects of incorporating other environmental, cellular, and chemical factors can be investigated.Item Dissolved oxygen and pH monitoring within cell culture media using a hydrogel microarray sensor(2009-05-15) Lee, Seung JoonProlonged exposure of humans and experimental animals to microgravity is known to be associated with a variety of physiological and cellular disturbances. With advancements in aerospace technology and prolonged space flights, both organism and cellular level understanding of the effects of microgravity on cells will become increasingly important in order to ensure the safety of prolonged space travel. To understand these effects at the cellular level, on-line sensor technology for the measurement and control of cell culture processes is required. To do this measurement, multiple sensors must be implemented to monitor various parameters of the cell culture medium. The model analytes used in this study were pH and dissolved oxygen which have physiological importance in a bioreactor environment. In most bioprocesses, pH and dissolved oxygen need to be monitored and controlled to maintain ionic strength and avoid hypoxia or hyperoxia. Current techniques used to monitor the value of these parameters within cell culture media are invasive and cannot be used to make on-line measurements in a closed-loop system. In this research, a microfabricated hydrogel microarray sensor was developed to monitor each anlyte. Either a pH or an oxygen sensitive fluorescent agent was immobilized into a hydrogel structure via a soft lithography technique and the intensity image of the sensor varied from the target analyte concentration. A compact detection system was developed to quantify concentration of each analyte based on the fluorescence image of the sensor. The system included a blue LED as an illumination source, coupling optics, interference filters and a compact moisture resistant CCD camera. Various tests were performed for the sensor (sensitivity, reversibility, and temporal/spatial uniformity) and the detection system (temporal/spatial stability for the light source and the detector). The detection system and the sensor were tested with a buffer solution and cell culture media off-line. The standard error of prediction for oxygen and pH detection was 0.7% and 0.1, respectively, and comparable to that of commercial probes, well within the range necessary for cell culture monitoring. Lastly, the system was coupled to a bioreactor and tested over 2 weeks. The sensitivity and stability of the system was affordable to monitor pH and dissolved oxygen and shows potential to be used for monitoring those analytes in cell culture media noninvasively.Item Electrokinetic concentration enrichment within a microfluidic device integrated with a hydrogel microplug(2009-05-15) Dhopeshwarkar, Rahul RajeshA simple and efficient technique for the concentration enrichment of charged species within a microfluidic device was developed. The functional component of the system is a hydrogel microplug photopolymerized inside the microfluidic channel. The fundamental properties of the nanoporous hydrogel microplug in modulating the electrokinetic transport during the concentration enrichment were investigated. The physicochemical properties of the hydrogel plug play a key role in determining the mode of concentration enrichment. A neutral hydrogel plug acts as a physical barrier to the electrophoretic transport of charged analytes resulting in size-based concentration enrichment. In contrast, an anionic hydrogel plug introduces concentration polarization effects, facilitating a size and charge-based concentration enrichment. The concentration polarization effects result in redistribution of the local electric field and subsequent lowering of the extent of concentration enrichment. In addition, an electroosmotic flow originating inside the pores of the anionic hydrogel manipulates the location of concentration enrichment. A theoretical model qualitatively consistent with the experimental observations is provided.Item Intein Engineering for Protien Hydrogel Synthesis and Protein Purification(2013-11-26) Ramirez, Miguel AngelInteins are proteins encoded within a precursor gene that excise themselves after translation and ligate the surrounding proteins with a peptide bond. Since their discovery two decades ago, many inteins have been engineered for various biotechnology applications. This dissertation focuses on the use and development of intein-based technologies for applications in protein purification and immobilization. The highly efficient naturally split DnaE intein from Nostoc punctiforme (Npu DnaE) was incorporated into synthetic protein building blocks for the synthesis of protein hydrogels, and engineered to catalyze rapid C-terminal cleavage reaction and used in the rapid purification of tag-less protein. In the first application, we developed protein hydrogels as general scaffolds for protein immobilization. Immobilization has been shown to increase protein stability and facilitate enzyme recovery-and-recycle tasks. These hydrogels are composed of artificial protein building-blocks expressed in bacterial hosts. Hydrogel gelation is catalyzed by intein-mediated protein trans-splicing reactions or disulfide bond formation between different protein building blocks. The resulting artificial protein hydrogels possess high solution stability at a wide range of pHs and temperatures, undergo shear-thinning, and are compatible with organic solvents. These self-assembled protein hydrogels can protect immobilized enzymes from organic solvent denaturation during biosynthesis, be used in enzymatic biofuel cells, and are suitable for the immobilization of multiple enzymes. In the second application, we engineered the Npu DnaE intein to catalyze rapid thio-induced C-terminal cleavage reaction and subsequently developed a split intein mediated technology for recombinant protein purification (SIRP). SIRP enables efficient purification of tag-less recombinant protein from E. coli lysate in less than 1 hour ? the hitherto fastest reported intein technology for protein purification.Item Novel In Situ-Gelling, Alginate-based Composites for Injectable Delivery: Tuning Mechanical and Functional Characteristics(2014-07-24) Roberts, Jason RichardThe development of fully-implantable therapeutic and diagnostic devices represents a new paradigm in biomedical device design. However, designing materials that can perform as injectable matrices for the delivery of sensing and therapeutics chemistries while retaining control over sensor and drug release behaviors is a complex problem. The novel material described herein, microporous alginate composite (MPAC), allows for controllable in situ gelation?and hence enables injection?as well as encapsulation of functional elements such as sensing chemistries or therapeutics. As this material has never been described before, individual component materials, bulk mechanical and gelation properties, and sensing composite response characteristics were examined. The use of polyelectrolyte multilayers (PEMs) in fabrication of MPACs resulted in a porous composite in which macromolecules and nanoparticles were retained within the pores, while allowing for free movement of these materials. Entrapped enzyme molecules were shown to react with diffusing substrates from outside the matrix, confirming the ability of materials from within the pores to interact with small molecules in the local environment. Increasing numbers of PEMs used in composite fabrication was found to result in increased gelation times of hydrogels, while increasing particle concentration reduced gelation times. Changes in pH during MPAC gelation was also dependent on microsphere concentration and PEM numbers. After gelation, MPAC hydrogels immersed in water displayed complex swelling and stiffening behavior dependent on particle concentration and PEM numbers. Oxygen-sensing MPAC hydrogels displayed minor PEM-dependent behavior, while glucose-sensing MPAC hydrogels displayed strong dependence on concentration and PEM numbers. As concentrations increased, sensitivities increased and analytical ranges decreased indicating cooperative behavior among enzyme-containing pores. Utilizing low permeability nanofilms, sensitivities and ranges of sensors could be modulated based upon the number of layers used in fabrication. The development of this new composite system architecture permits an added level of control over injectable hydrogel physical and functional properties such as gelation time and sensor response characteristics. This added control could broaden the usage of alginate as an injectable material and lead to the development of a wide variety of new functional injectable devices.Item Progress toward a Colon Targeting Nanoparticle Based Drug Delivery System(2012-07-16) Yu, XiaoHydrophobic drug paclitaxel nanoparticles (PAX NPs) and pH sensitive hydrogels were prepared in this study to build a colon targeting nanoparticle based drug delivery system for oral administration. Negative charged PAX NPs at the size of 110 +/- 10 nm were fabricated, characterized and then encapsulated in synthetic / biomacromolecule shell chitosan, dextran-sulfate using a layer by layer (LbL) self-assembly technique. Surface modifications were performed by covalently conjugating with poly (ethylene glycol) (H2N-PEG-carboxymethyl, Mw 3400) and fluorescence labeled wheat germ agglutinin (F-WGA), so as to build a biocompatible and targeted drug delivery system. Extended release of drug paclitaxel can be realized by adding more polyelectrolyte layers in the shell. High cell viability with PEG conjugated and high binding capacities of WGA modified nanoparticles with Caco-2 cells were observed. Preliminary study on stability of the nanoparticles in suspension at different pH was also performed. Two dextran based pH sensitive and enzyme degradable hydrogels: dextran maleic acid (Dex-MA), and glycidyl methacrylated dextran (Dex-GMA) were synthesized for oral delivery of nanoparticles. Hydrogels of both kinds were stable in simulated gastric fluid, but were prone to swelling and degradation in the presence or absence of enzyme dextranase in simulated intestinal fluid. The release profiles of nanoparticles could be tuned from 5 hr to 24 hr periods of time with more than 85% of the nanoparticle released in the simulated intestinal fluid. The release of PAX NPs was completed with longer time periods (45 hr-120 hr). Two possible release mechanisms were discussed for Dex-MA and Dex-GMA-co-AA hydrogels respectively: degradation controlled, and diffusion controlled. These biodegradable hydrogels, which can release nanoparticles depending on pH changes, together with the biocompatible and targeted nanoparticles, may be suitable as a potential colon targeting system for oral delivery of drug nanoparticles.