During wintertime temperature inversion episodes the concentrations of particulate air pollution, also defined as particulate matter (PM), in Utah’s Cache Valley have often been highest in the nation, with concentrations surpassing more populated and industrial areas. This has attracted much local and national attention to the area and its pollution. The Cache Valley has recently been declared to be in non-attainment of provisions of Federal law bringing to bear Federal regulatory attention as well. While there is epidemiological evidence indicating that PM is detrimental to public health, there is much less information indicating by which biological and molecular mechanisms PM can exert harm. This study was undertaken to better understand the mechanisms by which ambient PM collected in the Cache Valley can be harmful to human lung cells. Cache Valley PM was found to be mildly cytotoxic only at concentrations that were much greater than physiologically achievable, and such concentrations were difficult to obtain with the limited amounts of captured ambient PM. The limited cytotoxicity was despite apparent PM-induced pro-apoptotic signaling such as caspase-3 upregulation, and activation of caspase-12 and calpain. Cache Valley PM was found to be stressful to cells, triggering endoplasmic reticulum stress and the unfolded protein response. Cache Valley PM was also found to be inflammogenic leading to activation of pro-inflammatory transcription factors, increases in the release of pro-inflammatory cytokines and chemokines, as well as the upregulation of the activating receptors of these cytokines. The proinflammatory effects and absence of apoptosis, despite pro-apoptotic signaling of the Cache Valley PM on human lung cells appeared to stem from increased activation of the central pro-growth protein Akt with subsequent inactivation of the tumor suppressor P-TEN. These findings have indicated novel mechanisms of PM-related cellular stress and inflammation contributing needed information on what may be underlying mechanisms of PM associcated illnesses.

Targeted delivery of drugs directly to the lung epithelium is a promising, though challenging, strategy for the treatment of diseases that affect the lung tissues, such as infections caused by cell-penetrating pathogens, cystic fibrosis, and cancer. With appropriate surface functionality, such as through the attachment of ligands that recognize receptors on cellular surfaces, particulate carriers show improved efficiency in penetrating cells in vitro. A useful class of ligands is produced by many natural human pathogens that infect the respiratory tract. A variety of phylogenetically distinct respiratory bacterial pathogens, such as Haemophilus influenzae, invade host cells in the upper airways by binding of the platelet-activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms. By expressing host carbohydrate structures, including phosphorylcholine (ChoP), as a terminal structure on the LOS, the bacteria exhibit molecular mimicry of the host and are able to evade the host immune system. The effectiveness of LOS to induce cellular uptake of the bacteria is dependent on the specific glycoform, with higher ChoP content inducing more bacterial adherance into the lung epithelial. These ligands naturally expressed on bacterial cell surfaces can be isolated and utilized as targeting ligands for delivery vehicles. The studies described in this thesis focus on the development of particulate drug carriers coated with LOS bacterial ligands to enhance the targeting and binding of the carriers to the lung epithelium. Three NTHi clinical isolates were screened to select the strain with the highest ChoP level, and NTHi 3198, an isolate from a patient with chronic obstructive pulmonary disease (COPD), was selected due to its high ChoP activity. LOS from NTHi 3198 was isolated from the bacterial cell membrane, and its activity verified using dot immunoblot and ELISA techniques. Particles (0.2 and 1 µm) composed of polystyrene or poly(lactic-co-glycolic acid) were passively coated with 0.005-50 µg/mL of the isolated LOS 3198 with or without gelatin, coated with gelatin alone, or left uncoated. The LOS coating on the particles was verified using either XPS or ELISA. The association of particles with human bronchial epithelial cells was investigated using two cell culture models, 16HBE14o- and Calu-3, as a function of particle concentration and incubation time. The expression of PAFR on both cells types was confirmed, though the expression of PAFR on 16HBE14o- cells was significantly greater than on Calu-3 cells. Enhancement of 0.2 µm particle-cell association was achieved through coating of the particles with LOS. However, no significant difference in particle-cell association was observed for the 1 µm particles based on particle coating. Control particles of 0.2 µm size, those coated with gelatin (with or without LOS) or uncoated, exhibited low cell binding with a maximum of about 10-18% of cells associated with particles. The ability of the LOS ligand to enhance particle-cell association was coating concentration dependent, with a low coating concentration of LOS having little effect on association, but a concentration 1000-fold higher causing a doubling of the percentage of cells associated with particles at 24 hours. This enhancement was attributed to increased cellular binding of the 0.2 µm particles to the cell surface by confocal microscopy, and was further increased by activating the PAFR prior to incubation with particles. These results suggest the potential application of LOS as a targeting ligand for lung epithelial cells, especially under conditions where PAFR has been activated, such as occurs in lungs infected with Haemophilus influenzae. A significant reduction in particle-cell association was observed when particles were incubated with Calu-3 cells due to the presence of mucus on the cellular surface. This suggests that further optimization of the drug carrier system is needed to efficiently overcome the mucosal fluids.