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Macrophages are a cornerstone in innate immunity, especially important in detecting and killing invading microorganisms. In tumor biology, the macrophages can contribute both to anti-tumor activity and tumor promotion depending on individual tumor microenvironment and therefore have a large impact on both tumor progression and prognosis. Two of the most important functions of macrophages are the ability to phagocytose microorganisms and then kill them through the respiratory burst. Phagocytosis activates the respiratory burst, but the more subtle interactions between these processes are less known. Since phagocytosis and reactive oxygen species production are two attributes that change between the classically and alternatively activated macrophages we decided to compare these two functions in macrophages. Activation of macrophages varies in terms of stimuli and effects. We specifically looked at macrophage activation by tumor cell lines and by chemical stimulation due to caffeine. We hypothesized that the level of oxidation would be directly linked to the level of phagocytosis. We assume that caffeine will increase activity in macrophages and that tumor cell co-incubation will decrease it. We found that there is a high correlation between the level of engulfment and level of respiratory burst. Chemical stimulation with caffeine can lower aggressiveness of macrophages at lower concentration, raise it at higher concentrations and eventually become toxic to the cell. Co-incubation with leukemic cell lines, as well with necrotic cells, affected an increase in aggressiveness.

Chitosan (CS) is a man-made sugar based biopolymer derived from chitin, the second most abundant natural polymer after cellulose. Chitin is sourced from crustacean species such as shrimps and crabs. The chemical structure of chitin contains N-Acetyl D-glucosamine monomer units which forms CS upon deacetylation. In CS, ?-(1-4) linked D-glucosamine units are randomly distributed. Approximately 75% - 80% sugar units contains primary amine groups in commercially available low molecular weight CS. Biodegradability, low toxicity, mucoadhesive and transfecting properties of CS polymer are attractive for applications as oral and nasal drug delivery systems. Chitosan polymer is water insoluble at neutral pH. To solubilize CS, dilute mineral acid (such as hydrochloric acid and nitric acid) or organic acid (such as acetic acid) is often used. CS contains both hydroxyl and primary amine groups in its structure. In acidic solution, the amine functional groups become protonated (positively charged). Positively charged CS remains stable only in low pH condition due to electrostatic repulsion of charged polymer segments. Therefore, by using a suitable anionic (negatively charged) cross-linker, stable CS particles (such as nanoparticles and microspheres) can be prepared. This is popularly known as ionic gelation method. Extensive studies have been done on the synthesis of drug loaded CS particles where particle integrity is maintained by ionic gelation using tripolyphosphate (TPP, an anionic cross-linker). Drug encapsulated CS-TPP composite particles are shown to maintain biodegradability and biocompatibility. The CS-TPP composite particles exhibits very limited dispersibility at neutral pH conditions specifically in neutral buffered conditions. A number of biomedical applications (including systemic drug formulations) however demands buffer-stable CS composite particles for achieving optimal therapeutic outcome. To overcome the above dispersibility issues, CS polymer and CS particles units have been chemically modified using water soluble motifs (such as water soluble polymer or ligands). This approach is very cumbersome and usually involves multiple purification steps. Chemical modification of natural CS chain introduces risks of compromising biodegradability and biocompatibility. Therefore, there is a strong need for developing a straightforward method of making water soluble CS and CS particles. Chapter 1 of this dissertation presents an overview of the CS polymer, various applications of CS polymers, methods of making CS polymers and CS particles, current limitations of synthesis methods for preparing stable chitosan particles at neutral pH conditions and finally delineates the scope of the proposed research work. Chapter 2 describes development of chemo-hydrothermal synthesis method for producing water soluble CS polymer and water dispersible CS composite particles. In this method, a chemical (depolymerizing agent) is used to treat CS polymer in a hydrothermal (high temperature and high pressure) condition. Two types of depolymerizing agents have been used, an inorganic acid (e.g. hydrochloric acid, HCl) and a bicarboxylic organic acid (e.g. tartaric acid, TA). In both cases, 100% depolymerized CS polymer was obtained. Chemical characteristics of the depolymerized CS were comparable to acid solubilized CS. CS polymer exhibits weak fluorescence. Interestingly, hydrothermally depolymerized CS shows strong fluorescence properties irrespective of the nature of depolymerizing agent used. TA not only depolymerized CS but also formed CS-TA composite particulate structures in solution via self-assembly. The CS-TA composite particles are stable in a wide pH range from 5 to 11. Detailed spectroscopic and microscopic studies have been done to understand the basic mechanism of particle formation and increase in fluorescence properties (i.e. structure-property relationship). Usefulness of CS-TA in solubilizing water-insoluble cargos (such as fluorescein isothiocyanate, FITC) has been demonstrated. Chapter 3 is focused on hydrothermal synthesis of mixed-valence copper (Cu) oxide loaded CS-TA composite particles and their characterization. Crystalline Cu oxide nanoparticles were coated with the CS-TA layer. Water dispersibility of Cu oxide greatly improved upon coating with CS-TA material. To demonstrate catalytic activity of Cu-oxide loaded CS-TA film in sequestering carbon dioxide (CO2), an electrochemical setup was used. Electrochemical reduction of CO2 was successfully demonstrated. It was observed that CS-TA environment not only maintained catalytic properties of Cu oxide but also allowed solution processing of Cu-oxide film onto the electrode surface. Chapter 4 discusses a convenient method of making monodispersed water dispersible Cu loaded chitosan nanoparticles (Cu-CS) using HCl depolymerized CS polymer. The purpose of this study was to investigate if there was any improvement in antibacterial properties of Cu-CS nanoparticles prepared using hydrothermally treated CS polymer. Interestingly, it was observed that the antibacterial efficacy of Cu was not compromised in Cu-CS nanoparticles. Moreover, the materials exhibited improvement in antibacterial efficacy against both Gram-negative and Gram-positive bacteria species. A plausible mechanism has been proposed to explain antibacterial results. Chapter 5 summarizes major findings of this dissertation research and presents future research directions.

Parkinson's disease (PD) is a neurodegenerative condition that affects millions of people worldwide. The exact etiology of PD is unknown. However, it is well established that environmental factors contribute to the onset of PD. In particular, chemicals such as the insecticide Rotenone have been shown to increase the death of dopaminergic (DA) neurons by increasing levels of reactive oxygen species (ROS). ROS such as hydrogen peroxide (H2O2) have been shown to be elevated above basal levels in PD patients. Currently, to measure H2O2 concentrations, a commercially available (Amplex® Red) fluorescent assay is used. However, the assay has limitations: it is not completely specific to hydrogen peroxide and can only measure extracellular ROS concentrations. This research focuses on testing an electrochemical sensor that uses cyclic voltammetry to quantitatively determine concentrations of H2O2 released from a cell culture. The sensor was first tested in normal cell culture conditions. Next, chemical interference was reduced and the sensor was optimized for accuracy by altering protein concentrations in the media. Finally, Rotenone was added to a cell culture to induce H2O2 production. Near real-time measurements of H2O2 were taken using the sensor and comparisons made to the fluorescent assay method. Overall, we are trying to determine if the electrochemical sensor can selectively and quantitatively measure H2O2 released from cells. Being able to track the production, migration and concentration of H2O2 in a cell can help researchers better understand its mechanism of action in cell death and oxidative damage, thus getting closer to finding a cure for PD.

Side effects of radiation therapy (RT) remain the most challenging issue for pancreatic cancer treatment. In this report we determined whether and how cerium oxide nanoparticles (CONPs) sensitize pancreatic cancer cells to RT. CONP pretreatment enhanced radiation-induced reactive oxygen species (ROS) production preferentially in acidic cell-free solutions as well as acidic human pancreatic cancer cells. In acidic environments, CONPs favor the scavenging of superoxide radical over the hydroxyl peroxide resulting in accumulation of the latter whereas in neutral pH CONPs scavenge both. CONP treatment prior to RT markedly potentiated the cancer cell apoptosis both in culture and in tumors and the inhibition of the pancreatic tumor growth without harming the normal tissues or host mice. Mechanistically, CONPs were not able to significantly impact RT-induced DNA damage in cancer cells, thereby ruling out sensitization through increased mitotic catastrophe. However, JNK activation, which is known to be a key driver of RT-induced apoptosis, was significantly upregulated by co-treatment with CONPs and RT in pancreatic cancer cells in vitro and human pancreatic tumors in nude mice in vivo compared to CONPs or RT treatment alone. Further, CONP-driven increase in RT-induced JNK activation was associated with marked increases in Caspase 3/7 activation, indicative of apoptosis. We have shown CONPs increase ROS production in cancer cells; ROS has been shown to drive the oxidation of thioredoxin (TRX) 1 which results in the activation of Apoptosis Signaling iv Kinase (ASK) 1. The dramatic increase in ASK1 activation following the co-treatment of pancreatic cancer cells with CONPs followed by RT in vitro suggests that increased the c-Jun terminal kinase (JNK) activation is the result of increased TRX1 oxidation. The ability of CONPs to sensitize pancreatic cancer cells to RT was mitigated when the TRX1 oxidation was prevented by mutagenesis of a cysteine residue, or the JNK activation was blocked by an inhibitor,. Additionally, angiogenesis in pancreatic tumors treated with CONPs and RT was significantly reduced compared to other treatment options. Taken together, these data demonstrate an important role and mechanisms for CONPs in specifically killing cancer cells and provide novel insight into the utilization of CONPs as a radiosensitizer and therapeutic agent for pancreatic cancer.

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