Date of Degree
Access restricted until 07/13/2018
PhD (Doctor of Philosophy)
Pharmaceutical Sciences and Experimental Therapeutics
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.
lipooligosaccharide, lung cells, nanoparticle, pharmacy, pulmonary delivery, targeted delivery
xv, 229 pages
Includes bibliographical references (pages 214-229).
Copyright © 2015 Mai H. Tu