The vasculature is a highly complex, hierarchical system that performs a variety of functions in both physiological and pathological contexts. To maintain tissue homeostasis for example, the endothelium which lines all vascular structures generates a semi-permeable barrier that controls the exchange of fluids, ions, and solutes between the blood and tissue. During phases of tissue growth and wound repair, the vasculature undergoes angiogenesis, the development of new blood vessels, to provide adequate oxygen and nutrients to the new and healing tissues. In pathological situations such as cancer, blood vessels have been demonstrated to support tumor growth and provide access to the circulatory system for metastatic progression. This dissertation focuses on elucidating new mechanisms that are involved in regulating these three dynamic functions of the vasculature. In Chapter 2, we discuss preliminary work connecting the Notch signaling pathway with the ability for endothelial cells to mechanically couple to their substrate, a property that is known to regulate endothelial barrier function. Using traditional methods in two-dimensional traction force microscopy, we observed reductions in traction stresses generated by endothelial monolayers treated with a Notch inhibitor. This was accompanied by a decrease in cell- matrix tethering through focal adhesions. In Chapter 3, we utilized an engineered model of angiogenesis to probe the role of endothelial cell contractility in the formation of new vascular sprouts. Through these studies, we established an essential role of non-muscle myosin II in maintaining multicellularity during sprout morphogenesis. And in Chapter 4, we described the adaptation of a cranial window model for studying melanoma brain metastases and demonstrated the utility of this system to monitor dynamic interactions between cancer cells and the brain vasculature. Together, the work in this dissertation provides new insights into and techniques for probing outstanding questions regarding various key functions of the vasculature. / 2021-02-28T00:00:00Z

Electrochemical biosensors provide high specificity of analyte detection in different body fluids. A potentiostat is the instrument used to control the applied potential and measure current flow in the electrochemical cell, where the analyte detection reaction occurs. Advances in electronics and microcontrollers have enabled such sophisticated instruments to be built at a very low cost with sufficient performance. We report the analysis, design, and prototyping of a low-cost 4-channel potentiostat that is fully integrated into one PCB connected to an Arduino Uno board, as an Arduino shield, which could be used for both biosensor development and applications. As an Arduino-based instrument, the MATLAB-based software of the potentiostat is relatively easily modified for different biosensor requirements, providing great versatility for end users. We believe that this design will be valuable for electrochemical biosensors researchers as well as students interested in electrochemistry. The reported low-cost architecture could also be adopted to create low-cost diagnostic instruments for resource-limited settings.

Using the Layer by Layer (LbL) technique combined with microcontact printing, novel pH sensors were developed in order to detect the extracellular pH (pHe) of the cancerous cell lines K562 and HeLa. Key to this process was the utilization of fluorescence microscopy which allowed for direct measurement of the fluorescence intensity observed from the pH sensors made possible by the integration of fluorescent molecules into the polymer layers, namely Fluorescein Isothiocyanate and Rhodamine Isothiocyanate. In this work eight pH sensor types were analyzed and statistically validated. By subjecting the pH sensors to the pH buffers, pH 5, pH 6, pH 7, and pH 8 a standard curve was also able to be developed. Results found that both Nine Layer pH Sensor formulations, with FITC as the pH sensitive fluorophore, showed significant differences between pH value sets, such as between pH 6 and pH 7 and between pH 7 and pH 8, at **p<0.01 using T-Test analysis. These Nine Layer pH Sensors were then deemed suitable for cell seeding analysis since cells are known to exhibit extracellular pH’s between the ranges of pH 6 and pH 8. Cell seeding analysis of pH sensors revealed that no significant difference occurred with either cell type used, K562 or HeLa. In this analysis, cells were cultured over top of the pH sensor and allowed to bind ~2 nm away from the pH sensing fluorescent layer. It was hoped that the proximity of the layer to the cell would allow for a comparison between cell bound fluorescent striped regions and non-cell bound fluorescent striped regions. It was theorized that these cell bound regions would exhibit dimmed fluorescent stripes directly underneath the cells as the non-cell bound regions would exhibit greater fluorescence intensity comparatively. These two cancer cell lines are known to exhibit slightly acidic extracellular pH’s in the range of pH 6.7 to pH 6.8 while being cultured in media with known pH values of pH 7.4 or greater. This difference in pH values would then theoretically result in fluorescence intensity differences on the fluorescent stripes. In analyzing the pH sensors, it was seen that they did not possess the sensitivity needed to detect pHe, as they simply were only able to detect the pH of the bulk fluid and not pH directly surrounding the cells. Any visible fluorescence intensity change resulted from cell autofluorescence. Future work would then center around reducing interference from cell autofluorescence as well as determining the cause for the lack of sensitivity. It seems that manipulation of the polymer layers would possibly allow for greater diffusion of cell metabolites through the polymer matrix thus resulting in greater access to the fluorophore. It also seems that FITC may not be an adequate fluorophore and more sensitive molecule may need to be selected. Key words: Layer by Layer, microcontact printing, extracellular pH (pHe), K562, HeLa, Fluorescein Isothiocyanate, Rhodamine Isothiocyanate, fluorophore, T-Test. / A Thesis submitted to the Department of Chemical and Biomedical Engineering in partial fulfillment of the requirements for the degree of Master of Science. / Summer Semester 2017. / May 22, 2017. / extracellular pH (pHe), Layer by Layer, microcontact printing / Includes bibliographical references. / Jingjiao Guan, Professor Directing Thesis; Bruce Locke, Committee Member; Ravindran Chella, Committee Member.

Recent advancements in multilayer, multicellular, genetic logic circuits often rely on manual intervention throughout the computation cycle and orthogonal signals for each chemical “wire”. These constraints can prevent genetic circuits from scaling. Microfluidic devices can be used to mitigate these constraints. However, continuous-flow microfluidics are largely designed through artisanal processes involving hand-drawing features and accomplishing design rule checks visually: processes that are also inextensible. Additionally, continuous-flow microfluidic routing is only a consideration during chip design and, once built, the routing structure becomes “frozen in silicon,” or for many microfluidic chips “frozen in polydimethylsiloxane (PDMS)”; any changes to fluid routing often require an entirely new device and control infrastructure. The cost of fabricating and controlling a new device is high in terms of time and money; attempts to reduce one cost measure are, generally, paid through increases in the other. This work has three main thrusts: to create a microfluidic fabrication framework, called MakerFluidics, that lowers the barrier to entry for designing and fabricating microfluidics in a manner amenable to automation; to prove this methodology can design, fabricate, and control complex and novel microfluidic devices; and to demonstrate the methodology can be used to solve biologically-relevant problems. Utilizing accessible technologies, rapid prototyping, and scalable design practices, the MakerFluidics framework has demonstrated its ability to design, fabricate and control novel, complex and scalable microfludic devices. This was proven through the development of a reconfigurable, continuous-flow routing fabric driven by a modular, scalable primitive called a transposer. In addition to creating complex microfluidic networks, MakerFluidics was deployed in support of cutting-edge, application-focused research at the Charles Stark Draper Laboratory. Informed by a design of experiments approach using the parametric rapid prototyping capabilities made possible by MakerFluidics, a plastic blood--bacteria separation device was optimized, demonstrating that the new device geometry can separate bacteria from blood while operating at 275% greater flow rate as well as reduce the power requirement by 82% for equivalent separation performance when compared to the state of the art. Ultimately, MakerFluidics demonstrated the ability to design, fabricate, and control complex and practical microfluidic devices while lowering the barrier to entry to continuous-flow microfluidics, thus democratizing cutting edge technology beyond a handful of well-resourced and specialized labs.

The first standardized methods for in vitro drug release testing of solid dosage forms were first introduced in the 1960s. Drug release testing has since become an important analytical measure along all stages of the drug development process. Despite the expanded role of dissolution testing and innovations in the types of dosage forms reaching the market, the fundamental methods and approaches to dissolution testing have not changed from their original introduction. This lack of innovation and one-size-fits-all approach to drug release testing has led to inefficiencies in testing and limited the scope of applications where this type of information could have an impact. In order to meet this need, we have designed, characterized, and implemented a small volume drug release test using ultrasonic agitation to screen for differences in dosage form composition. Our approach aims to supplement official methods for use during multiple stages of the drug development process. The hydro-acoustic environment in the system was characterized as a function of input power and position of the acoustic source. Drug release behavior from tablets was also studied over these system parameters, and a preliminary mechanistic explanation is made linking the two. The interplay between fragmentation and diffusion on solid dissolution processes was then explored through a deterministic partial differential equation model. This model provides the first instance of time-evolving particle size distributions in a dissolution model. In the final sections of this dissertation, uses of the ultrasonic agitation mediated drug screening method are demonstrated at two different parts of the drug development process – during early formulation development for the study of composite microparticle matrix structure on drug release behavior and post market surveillance for the screening of substandard tablets. / 2020-02-28T00:00:00Z

Sarcomas are broadly defined as cancers that form in the bone, soft tissue, or connective tissue. While they represent only 1% of all cancers in the United States, sarcomas constitute 12% of all childhood cancers. Five-year survival has not changed in over 40 years. The only clinically accepted indicator of pathologic response and disease-free survival is percent tumor-cell necrosis at time of surgery---there are no established prognostic markers before surgery. Unfortunately, 40--70% of patients have a poor pathologic response and attempts to modify treatment to improve their outcomes have been unsuccessful. Diffuse Optical Spectroscopic Imaging (DOSI) is a non-invasive, functional imagining technique that has been previously implemented to predict pathologic response in patients with breast cancer. Specifically, DOSI combines frequency amplitude modulated near-infrared light and broadband continuous wave light to measure quantitative concentrations of oxyhemoglobin, deoxyhemoglobin, water, and lipids. This project developed and validated DOSI as a new non-invasive measurement modality to track treatment for sarcomas. For the first time, the optical properties and functional hemodynamic information of the distal femur, tibia, and humerus were characterized in normal volunteers. DOSI demonstrated the ability to measure optical properties and functional information at several sarcoma locations throughout the course of treatment. It was able to differentiate between healthy and sarcoma tissues within a patient. Improvements made to the instrumentation will facilitate future measurements in this patient population. In the future, DOSI may provide a way to monitor treatment response and improve patient outcomes. / 2019-12-04T00:00:00Z

The formulation of hydrophobic drugs into appropriate dosage forms is challenging due to the problems associated with those drugs such as low solubility and poor dissolution. Using a liquisolid system is a promising method to improve the dissolution of hydrophobic drugs and in sustaining the release of hydrophilic drugs, in which solid drugs are dispersed in non-volatile liquid vehicles. The aim of this research was to use the liquisolid technique to enhance the dissolution rate of glibenclamide, a model hydrophobic drug, and to sustain the release of metformin-HCl, as a model hydrophilic drug. The wet granulation process was applied to liquisolid powders with the aim of overcoming issues of poor powder flowability and compressibility, especially using high viscosity liquid vehicles. This process was performed with liquisolid powders prior to compaction into tablets. Different liquisolid formulations were prepared using three liquid vehicles (polyethylene glycol400 (PEG® 400), Synperonic® PE/L44 and Cremophor® ELP), at 10 and 30 % w/w drug concentrations for glibenclamide; and 30% and 60% w/w drug concentrations for metformin-HCl. Avicel®PH102 was used as a carrier, whilst colloidal silicon dioxide was employed as a coating material to convert the wet mixtures into dry powders. Potato starch, 5% w/w, as a disintegrant was blended with the mixtures manually for 10 minutes and then 0.75% of magnesium stearate as a lubricant was added and mixed for 5 minutes. The final powder (depending on its flowability and compactability) was then compacted automatically using a single-punch tableting machine to give tablets with 4 mg for glibenclamide and 40 mg for metformin-HCl. Prepared liquisolid compacts were characterized by using British Pharmacopeia quality control tests: uniformity of weight, friability, disintegration, hardness and drug dissolution. iii It was found, for both drugs, that by application of wet granulation to liquisolid powder admixtures, the large-scale production of liquisolid compacts is feasible, which can be easily adapted to the pharmaceutical industry. In addition to enhancing the flowability and compressibility of the powders, the glibenclamide dissolution was also improved due to the enhanced binding of particles and because of the wetting effect of liquid vehicles on the hydrophobic drug, which make the drug more available for dissolution. For the sustained release preparations of liquisolid metformin-HCl, hydroxyl propyl cellulose (HPC) was used as a novel carrier in liquisolid compacts. The results showed 92% drug release after 12 hours using Cremophor®ELP (with 30% w/w drug concentration) which was the best sustained drug release formulation. Additionally, Eudragit® RL30D and Eudragit® RLPO have been used to study their effects on drug release from liquisolid formulations, examining if they can sustain or give more rapid drug release. Both types of Eudragit revealed immediate release with metformin-HCl rather than sustained drug release, with the tablets disintegrating within seconds. This suggests formulating orodispersible metformin-HCl tablets using Eudragit® RL30D as a liquid vehicle. In summary, liquisolid technology has led to promising results, not only in enhancing the drug dissolution of hydrophobic drugs, but also in sustaining and promoting the release of hydrophilic drugs.

The current study has focused on the local delivery of furazolidone to the gut with the aim of generating alternative approaches for the treatment of Helicobacter pylori (H. pylori). Furazolidone has proven antibacterial activity against H. pylori, which has a unique niche in the stomach mucus. This drug was chosen as it is not currently used for the treatment of H. pylori and thus resistance is not expected to be a problem. Chitosan micro-particles were formulated by the spray drying technique, followed by optimization of mucoadhesion and drug release profiles using glutaraldehyde crosslinking agent at two pH values (1.3 and 4.5). Results revealed that increasing glutaraldehyde decreased the mucin adsorption and at low pH drug release was increased. For liposomal formulations, the effects of furazolidone concentration, chitosan and cholesterol on encapsulation efficacy and in vitro drug release were evaluated. It was found that increasing the pH from 1.3 to 4.5 increased the mucoadhesive behaviour of chitosan coated liposomes from 42% to 60%. Also, increasing the furazolidone amount from 4mg to 5mg increased encapsulation efficiency. A combination of two antibiotics (including furazolidone) was prepared in muco-penetrative liposomal formulations; N-acetylcysteine was used for the muco- penetration effect with Pluronic F-127. These formulations were investigated for their charge effect on muco-penetration and drug encapsulation. The data showed that neutral liposomes easily diffuse through the mucus layer. Escherichia coli was selected to establish the assay protocol for Helicobacter pylori. The microdilution approach was used for assaying the furazolidone minimum inhibitory concentration (MIC), which was found to be 16 μg/ml for E. coli and 4 μg/ml for H. pylori. In time-dependent killing studies, it was possible to observe iii complete killing of the bacteria. Increasing furazolidone concentration by two fold of its MIC, reduced the time required to kill bacteria. The mucoadhesive drug formulations also increased the residence time of furazolidone in the stomach mucus from 2- 3 hours to 4-6 hours; this time period would then be appropriate for killing the bacteria in the stomach. For mucopenetration study complete killing was achieved in 2.5 hours when furazolidone with 1 % minimum inhibitory concentration of NAC which was used. Which was otherwise six hours when NAC was not added for augmentation. To conclude, delivery of furazolidone was via application of novel liposomal and spray dried formulations to either increase movement across gastric mucosa (via a muco-penetration effect) or to increase binding to the mucus (via mucoadhesive action). Hence, the various approaches used in this research have showed success (to deliver effective amounts of furazolidone locally into the stomach mucus) and the co-encapsulation of furazolidone and N-acetylcysteine is a novel approach for the delivery of antimicrobial agents to the stomach.

Smooth muscle cells exist in many different locations within the body, including blood vessels and airways, where their principal function is contraction and relaxation. The heterogeneity of smooth muscle cells has been related to their embryological origins and could have implications in many diseases, including atherosclerosis, pulmonary hypertension, and asthma. Many of these diseases require an expandable cell source of smooth muscle cells for regenerative medicine or disease modeling. Here, we have developed Acta2hrGFP and ACTA2eGFP (GFP reporters for smooth muscle α-actin) reporter mouse and human induced pluripotent stem cells lines to track and isolate populations of smooth muscle-like cells. iPSCs were patterned to a KDR-expressing (kinase insert domain receptor) mesodermal progenitor, which was further specified towards a smooth muscle-like lineage through exposure to platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-β). The Acta2hrGFP+ or ACTA2eGFP+ cells were enriched for characteristic markers of smooth muscle cells, and these cells expressed low levels of contractile markers, reminiscent of an immature or synthetic smooth muscle cell. Aligned smooth muscle-like cell sheets were generated using these iPSC-derived populations in an enzymatically degradable hydrogel system. The cell sheets displayed mechanical behavior similar to native blood vessels, with the Acta2hrGFP+ cell sheets displaying a higher ultimate tensile strength than Acta2hrGFP- cell sheets. Furthermore, we performed global transcriptomic profiling of primary adult mouse lung vascular (Acta2hrGFP+ Cspg4DsRed+) and airway (Acta2hrGFP+ Cspg4DsRed-) smooth muscle cells from a double transgenic reporter mouse, where we identified distinct gene signatures of lung vascular SMCs and airway SMCs, with Hhip and Acta2 co-expression distinguishing airway SMCs from lung vascular SMCs. When comparing our miPSC-derived Acta2hrGFP+ cells to these primary SMC signatures, the in vitro derived cells cluster closer to aortic SMCs and lung vascular SMCs, but their transcriptomic signatures still remain significantly distinct. In addition, we have generated an Acta2hrGFP Cspg4DsRed reporter mouse iPSC line, which can be used to understand the signaling pathways involved in specification of these different smooth muscle cell subtypes. Thus, we have developed systems for isolating smooth muscle-like populations which have potential in tissue engineering applications, and we have identified gene signatures of adult lung vascular and airway smooth muscle cells to begin to address the heterogeneity of smooth muscle cell lineages.

<p> Natural materials have been processed and utilized as scaffold materials in the field of tissue engineering for many years. One natural material often utilized is collagen since it is the main structural protein in mammalian tissues and exhibits microstructures suitable for the survival and proliferation of many different cell lineages. However, a common challenge with fibrillized collagen is the difficulty associated with trying to process it into specific three-dimensional designs for the development of scaffolds aimed at regenerating particular tissue types. This project consists of utilizing a custom platform capable of 3D printing <i>in situ</i> polymerizing collagen into user-defined morphologies for the development of 3D collagen-based scaffolds. Various anti-inflammatory compounds such as gold nanoparticles and curcumin were also incorporated into the scaffolds post printing in order to further tailor the cellular responses to the scaffolds. Scanning electron microscopy and neutron activation analysis were performed to verify and quantify the attachment of the gold nanoparticles, respectively. Differential scanning calorimetry was utilized to examine and optimize the stability of the scaffolds after crosslinking. Lastly, water soluble tetrazolium salt and reactive oxygen species assays were performed to assess the biocompatibility of the scaffolds using L929 murine fibroblasts. The results exhibited the viability of the platform to become an effective technique to manufacture and process custom scaffolds for tissue engineering applications.</p><p>