Chemical and Biomolecular Engineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2751
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Item The Delivery of IGF-1 for Skeletal Muscle Regeneration Within Abdominal Wall Hernia Repair(2009) Falco, Erin Elizabeth; Fisher, John P.; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)At an ever increasing pace, synthetic biomaterials are being developed with specific functionalities for tissue engineering applications. These biomaterials possess properties including biocompatibility, mechanical strength, and degradation as well as functionalities such as specific cell adhesion and directed cell migration. However, synthetic polymers are often not completely biologically inert and may non-specifically react with the surrounding in vivo environment. An example of this reactivity is the release of acidic degradation products from hydrolytically degradable polymers based upon an ester moiety. These degradation products can lower the local pH and incite an inflammatory response as well as increase scaffold degradation rate. Therefore there has been a concerted effort in the research community to develop alternatives. In order to address this concern, a novel class of biomaterials based upon a cyclic acetal unit has been developed and investigated for both soft and hard tissue repair. This work specifically looks at a cyclic acetal biomaterial based on a 5-ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ethanol diacrylate (EHD) monomer as a scaffold for abdominal wall hernia repair. Abdominal wall hernias are a growing concern for clinicians today as they occur in approximately 10% of all patients that undergo an abdominal procedure. Despite many advances in repair techniques, both wound healing and skeletal muscle regeneration is limited in many cases. This results in both a decrease in abdominal wall tissue function as well as a hernia recurrence rate of up to 50%. To address this high recurrence rate this project aims to create a functional gene delivery scaffold from the EH monomer. Scaffolds with different architectures were fabricated and skeletal muscle myoblast cell compatibility, material properties and protein and gene delivery rates were all investigated.Item IN-SITU MEASUREMENT OF EPITHELIAL TISSUE OPTICAL PROPERTIES: DEVELOPMENT AND IMPLEMENTATION OF DIFFUSE REFLECTANCE SPECTROSCOPY TECHNIQUES(2009) Wang, Quanzeng; Wang, Nam Sun; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cancer is a severe threat to human health. Early detection is considered the best way to increase the chance for survival. While the traditional cancer detection method, biopsy, is invasive, noninvasive optical diagnostic techniques are revolutionizing the way that cancer is diagnosed. Reflectance spectroscopy is one of these optical spectroscopy techniques showing promise as a diagnostic tool for pre-cancer detection. When a neoplasia occurs in tissue, morphologic and biochemical changes happen in the tissue, which in turn results in the change of optical properties and reflectance spectroscopy. Therefore, a pre-cancer can be detected by extracting optical properties from reflectance spectroscopy. This dissertation described the construction of a fiberoptic based reflectance system and the development of a series of modeling studies. This research is aimed at establishing an improved understanding of the optical properties of mucosal tissues by analyzing reflectance signals at different wavelengths. The ultimate goal is to reveal the potential of reflectance-based optical diagnosis of pre-cancer. The research is detailed in Chapter 3 through Chapter 5. Although related with each other, each chapter was designed to become a journal paper ultimately. In Chapter 3, a multi-wavelength, fiberoptic system was constructed, evaluated and implemented to determine internal tissue optical properties at ultraviolet A and visible wavelengths. A condensed Monte Carlo model was deployed to simulate light-tissue interaction and generate spatially distributed reflectance data. These data were used to train an inverse neural network model to extract tissue optical properties from reflectance. Optical properties of porcine mucosal and liver tissues were finally measured. In Chapter 4, the condensed Monte Carlo method was extended so that it can rapidly simulate reflectance from a single illumination-detection fiber thus enabling the calculation of large data sets. The model was implemented to study spectral reflectance changes due to breast cancer. The effect of adding an illumination-detection fiber to a linear array fiber for optical property determination was also evaluated. In Chapter 5, an investigation of extracting the optical properties from two-layer tissues was performed. The relationship between spatially-resolved reflectance distributions and optical properties in two-layer tissue was investigated. Based on all the aforementioned studies, spatially resolved reflectance system coupled with condensed Monte Carlo and neural network models was found to be objective and appear to be sensitive and accurate in quantitatively assessing optical property change of mucosal tissues.Item Insulin-Like Growth Factor-1 Signaling in Engineered Articular Cartilage(2008-11-24) Yoon, Diana Meeae; Fisher, John P; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Articular cartilage can easily become damaged or diseased and it does not have the ability to heal itself. A tissue engineering approach to regenerate cartilage is to integrate chondrocytes, the primary cell in cartilage, with biomaterials and biomolecules. Currently, there is limited knowledge on how all these factors influence the expression of upstream insulin-like growth factor-1 (IGF-1) signaling molecules. In an effort to better understand how IGF-1 and phenotypic, type II collagen, expression can be modified by altering construct properties, chondrocytes were embedded in alginate hydrogels. Increasing alginate concentration resulted in an upregulation of IGF-1 expression and by increasing cell density further enhanced IGF-1 expression. Additional changes in chondrocyte signaling were observed when exogenously delivering IGF-1 to the constructs. IGF-1 expression decreased while the receptor for IGF-1 (IGF-1R) expression as well as type II collagen increased in the presence of excess IGF-1 indicating that it has a key role in IGF-1 and chondrocyte interaction. An extracellular matrix molecule, such as hyaluronic acid (HA), provides anchorage sites for chondrocytes and therefore the influence of HA on IGF-1 signaling was also investigated. The incorporation of HA created a dual affect by entrapping exogenously delivered IGF-1 as well as directly interacting with chondrocytes. As a result, the IGF-1 expression levels varied depending on HA concentration and there was also a lack of correlation with IGF-1R expression. The upregulated expression of type I collagen, a fibroblastic marker, by chondrocytes indicated that HA can overcome the beneficial affects of IGF-1. These in vitro works were also compared to an in vivo study. Alginate/HA constructs with embedded chondrocytes were pre-cultured with IGF-1 and then subcutaneously implanted into mice. Similar levels of type II collagen were observed for the constructs. However, by increasing the HA content a decrease in IGF-1 synthesis occurred with an increase in type I collagen staining. Pre-incubating the samples with IGF-1 led to a further decrease in IGF-1 production but was able to reverse the affects of HA on type I collagen expression. This research demonstrates that construct properties can alter endogenous IGF-1 signaling and overall shows the importance of understanding these details when engineering an articular cartilage construct.Item RNA packaging and gene delivery using Tobacco mosaic virus pseudo virions(2008-04-28) Hung, Chi-Wei; Bentley, William; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA interference (RNAi) has emerged as a powerful tool for the study of gene function and post-transcriptional regulation. However, the lack of a proper delivery system for RNAi is a major problem for its application as a therapeutic agent. In this study, Tobacco mosaic virus (TMV) is utilized as an RNAi carrier for gene delivery into mammalian and insect cells. The self assembly and disassembly of TMV is investigated to create chimeric viruses for gene delivery. The origin of assembly sequence (OAS) within the TMV RNA initiates its association with coat protein through a unique hairpin structure. Studies in this dissertation show that by incorporating TMV OAS into an RNA of interest, the RNA can assemble into "pseudo-virions" by the virus coat protein. The length of the pseudo-virions changed in proportion with the size of the RNA. To deliver the RNA to the targeted cells, virions are further surface-modified with synthetic cell-penetrating peptides to facilitate cell endocytosis. Two genes were selected as targets: 1) EGFP as a visual marker and 2) Cyclin E for control of cell cycle. EGFP is expressed in a transient expression experiment using a plasmid vector, pEFGP-N1. Cyclin E is regulated endogenously in High FiveTM cells, and its translation is targeted using the pseudo virions. Pseudo-virions targeting EGFP RNA (antisense EGFP) are able to suppress transient EGFP production by 61% whereas pseudo virions targeting cyclin E (antisense cycE) are capable of arresting cells at G1 phase. This RNA packaging system protects packaged RNA and provides a means of delivering RNAi constructs into various host cells.Item Analysis of Drug Delivery in the Eye Using Magnetic Resonance Imaging(2007-11-19) Kim, Heekyong Stephanie; Wang, Nam S; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)With the rapid increase in the elderly population, the number of Americans afflicted with vision impairment due to ocular disease is projected to rise substantially by the year 2020. Ocular disorders are becoming a major public health problem, and efforts have increased in recent years to develop methods of efficient drug delivery. Currently, the most effective method for treating serious ocular disorders is to inject drug solutions directly into the vitreous. However, injecting in this manner carries a high risk of severe side effects. As a safer alternative, researchers in recent years have been investigating transscleral drug delivery, in which the drug is administered to the outer coat of the eye. Various methods of transscleral drug delivery have been proposed, but it is still clinically not as effective as intravitreal drug delivery. In order to design improved transscleral drug delivery systems, the ocular barriers to drug transport must be accurately understood. While various barrier types have been identified in the eye, the significance and contribution of individual barriers have not been investigated and are still widely unknown. A reason for this lack of understanding is due to the inability to acquire concentration measurements in the eye in vivo. In this study, magnetic resonance imaging (MRI) was employed to obtain drug concentration measurements in vivo after transscleral drug delivery. To address the current needs of the ocular drug delivery community, several goals have been achieved in this work: (1) to evaluate transscleral drug delivery in vivo using MRI, (2) to assess MRI as a technique for evaluating drug delivery in the eye, and (3) to better understand the significance of individual barriers in the eye by quantitatively analyzing experimental (MRI) data and by pharmacokinetic modeling. While encompassing many advantages, it is found that MRI has limitations in spatial and temporal resolution that may restrict its use in measuring parameters with low sensitivity. However, the MRI results in parallel with analysis from the pharmacokinetic model give new insight into the barriers to drug transport in the eye.Item Rewiring Quorum Sensing Circuitry for Recombinant Protein Production in E. coi(2007-08-08) Tsao, Chen-Yu; Bentley, William E.; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The global objective of this research is to rewire the circuitry of bacterial quorum sensing to facilitate recombinant protein production in bacteria. Previous research has shown that the activity of AI-2, the putative "universal" bacterial autoinducer, decreased in culture fluids when several proteins were overexpressed in E. coli W3110, suggesting bacteria communicate or possibly potentiate the "metabolic burden" associated with protein overexpression. Additionally, conditioned medium obtained from LuxS+ and LuxS- strains was added to these cultures, resulting in a 2-4 fold increase in specific yield for both chloramphenicol acetyltransferase (CAT) and organophosphorus hydrolase (OPH). These simple observations set the stage for examining the role of quorum sensing in recombinant protein expression systems and also suggested that "rewiring" the quorum sensing circuitry would lead to significant improvement of yield. In this dissertation, we have inserted luxS into expression vectors (IPTG inducible) which can co-synthesize target recombinant proteins (arabinose inducible) to accomplish the modulation of the metabolic landscape for protein synthesis via altered AI-2 signaling. Our results show significant enhancement in both protein yield and activity, and reveal a strong linkage between bacterial cell communication and cellular processes involved in synthesis and folding of recombinant proteins in E. coli. Second, we have attempted to rewire the native quorum sensing signaling circuitry and couple it to the widely-used T7 expression system for constructing an autoinducible recombinant protein expression platform. We demonstrate, for the first time, true autoinduction of recombinant proteins in E. coli or, in fact, any expression system. That is, our results showed that GFPuv, CAT, and LacZ were all expressed using this innovative system without cell growth monitoring or inducer addition.Item Molecularly Imprinted Polymers for the Recognition of Tobacco Viruses(2007-07-31) Bolisay, Linden De Venecia; Kofinas, Peter; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The goal of this research is to elucidate the mechanism of virus recognition in molecularly imprinted polymers (MIPs) using already utilized techniques. The clinical relevance of this study relates to the development of a virus imprinted MIP, which would apply to the identification, classification, and removal of viruses. The separation of viruses and virus-like particles from various media represents an enormous challenge to the fields of medicine, healthcare, and biotechnology. Since virus MIPs must function in aqueous environments, our approach employs a more flexible non-covalent imprinting method which starts from a readily available polymer and utilizes an aqueous environment for both MIP synthesis and testing. Crosslinked polymers imprinted against Tobacco mosaic virus (TMV) via non-covalent interactions were synthesized using poly (allylamine hydrochloride) (PAA), epichlorohydrin (EPI), and TMV. The TMV imprinted polymer exhibited an increase affinity to the target virus compared to the control polymer and demonstrated a preferential affinity (imprinting factor of 2.1), based on shape, to the target virus compared to a non-target virus, Tobacco necrosis virus (TNV). In contrast, there was no significant increase in binding of the control polymer to either target or non-target virus. Once it was determined that virus imprinted polymers can be successfully synthesized having preferential binding to a targeted virus, the synthesis procedure was optimized to obtain better binding characteristics to the targeted virus. Efforts were made to avoid polymer-template aggregation in the MIP pre-polymerization mixture, and determine a proper wash solution by the ability to remove the templated virus from the crosslinked polymer. TMV imprinted hydrogels were synthesized using an optimized procedure and binding test performed on these MIPs to determine binding capacity, and more importantly, imprinting factor. The highest imprinting factor of 2.3 resulted from the MIP composed of 35 % PAA at pH 7, 15 %, ethylene glycol diglycidyl ether (EGDE), and 0.4 mg/mL TMV. The TMV imprinted hydrogels exhibited a lower binding capacity to TNV than when exposed to TMV. These results show that using optimized procedures, TMV MIPs with better shape selectivity can be achieved.Item SYSTEMATIC INVESTIGATION OF QUORUM SENSING IN Escherichia coli(2007-04-17) Li, Jun; Bentley, William E; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)High throughput techniques and advanced mathematical tools have enabled systematic investigations of biological systems with unparalleled precision. Not only molecular interactions between components but mechanisms and the dynamic behaviors associated with these systems are revealed, suggesting that comprehensive systems biology can be realized in the near future. Quorum sensing, especially the auto-inducer2 (AI-2) system, has been extensively studied due to its commonality among bacteria and connections to pathogenic phenotypes. In this study, the E. coli quorum sensing AI-2 system was studied combing system-based mathematical modeling and high throughput genomic profiling. First, a Stochastic Petri Network (SPN) model was constructed based on available regulatory information. Simulations together with experimental data demonstrated that the apparent stimulation of AI-2 in the presence of glucose is not from the increased transcriptional or translational expression of AI-2 synthases luxS and pfs, nor from the increased metabolic flux associated with LuxS-related pathways but from an alternative AI-2 synthesis pathway. The conversion of adenosine with cellular extracts from both luxS and pfs mutants validated our prediction about the existence of an alternative non-LuxS related AI-2 synthesis pathway. Second, AI-2 uptake regulatory network was investigated in detail: lsrR-lacZ, lsrK-lacZ fusion reporters were constructed and the analysis found that lsrR is subject to its own repression and is induced by both lsrK and luxS. Further transcriptome analysis demonstrated that lsrR and lsrK, together with quorum signal AI-2, coregulate lsrRK regulon, which influences phenotypes (biofilm, small RNAs). Importantly, this regulation is in a distinctly different manner than that mediating the lsr operon. We hypothesize that lsrR acts together with AI-2 to mediate cellular processes and that the phosphorylation of AI-2 molecule through lsrK triggers different response pathways. These investigations demonstrated that lsrR, lsrK are indispensable for AI-2 uptake. These newly elucidated regulatory mechanisms and associations undoubtedly broaden the scope of the AI-2 quorum sensing system, and provide a solid foundation for further mathematical modeling of the dynamics and system behaviors in E. coli . Finally, a tight coupling of experimental manipulation with mathematical analysis, as demonstrated in this study, provides a good example for systematically investigating biological systems.Item Study of ocular transport of drugs released from a sustained release device(2004-05-03) Kim, Hyuncheol; Wang, Nam Sun; Chemical EngineeringDelivering ocular therapeutics to a target site with minimal side effects requires detailed information about the distribution and elimination pathways. This knowledge can guide the development of new drug delivery devices. In this study, we investigated the movement of two drug surrogates, H-110, which is lipophilic, and Gd-DTPA, which is hydrophilic, released from polymer-based implants using a fluorescein technique and magnetic resonance imaging (MRI). We also studied the pharmacokinetics of intravitreally injected triamcinolone acetonide, a low water soluble drug used for treating sight-threatening diseases such as diabetic retinopathy and choroidal neovascularization associated with age-related macular degeneration (AMD). At 24 hour post implantation, H-110 released from an intravitreal implant was detected in the subretinal space. However, following a subconjunctival implant, very little H-110 fluorescence was detected in the subretinal region. H-110 most likely reached the subretinal space from an intravitreal implant by diffusion through the vitreous and retina. However, most of the H-110 released from a subconjunctival implant is thought to dissipate through the choroidal blood flow. Concentration profiles of Gd-DTPA, which was released from an intravitreal implant in a New Zealand white rabbit, approached pseudo-steady state within 7 to 8 hours and showed gradients at the rabbit's vitreous-retina border suggesting that diffusion was occurring into the retinal-choroidal-scleral membrane. Parametric analysis with a finite element mathematical model of the rabbit eye yielded for Gd-DTPA a diffusion coefficient of 2.8 × 10-6 cm2/sec in the vitreous and a permeability of 1.0 × 10-5 cm/sec in the composite retina-choroid-sclera membrane. Gd-DTPA concentration decreased away from the implant. Such regional concentration variations throughout the vitreous may have clinical significance when the ubiquitous eye diseases are treated using a single positional implant. Subconjunctival implants in vivo delivered a mean total of 2.7 µg of Gd-DTPA over 8 hours into the vitreous representing only 0.12% of the total amount of compound released from the implant in vitro. No Gd-DTPA was detected in the posterior segment of the eye. Ex vivo, the Gd-DTPA concentration in the vitreous was 30 fold higher suggesting the elimination of significant in vivo barriers to the movement of drugs from the subconjunctival space into the vitreous. We developed a new preservative-free formulation for intravitreal injections of triamcinolone acetonide for the treatment of diabetic macular edema, and choroidal neovascularization associated with AMD in human clinical trials at the National Institutes of Health. A pharmacokinetic study in rabbits was done to estimate elimination rate of two injection amounts of triamcinolone acetonide, 4 mg and 16 mg, from the vitreous. From our pharmacokinetic model, we found the half-lives for 4 mg and 16 mg injection in the vitreous to be 18.6 days and 37.6 days, respectively. We subsequently estimated the half-lives of 1 mg and 8 mg triamcinolone acetonide injection in order to predict therapeutic exposure in human. There are three components in this thesis: the study of lipophilic H-110 transport with fluorescence, the study of hydrophilic transport of Gd-DTPA with MRI, and the pharmacokinetic analysis of triamcinolone acetonide. They have each contributed to further insights into our fundamental understanding of drug movement in the eye and the implication on optimal therapeutic delivery.