CHARACTERIZATION OF BACTERIOPHAGE AND MACROPHAGE INTERACTIONS FOR APPLICATIONS IN PULMONARY DRUG DELIVERY
Date
2025-12
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Publisher
University of Delaware
Abstract
Lower respiratory infections (LRIs) remain a leading global health burden, with bacterial infiltration and biofilms that can limit antibiotic efficacy. Bacteriophages are a promising alternative to antibiotics, capable of lysing bacterial pathogens. Their nanoscale size enables deposition in the lower respiratory tract and interaction with innate immune cells. However, the development of inhalable phage therapies is underexplored due to a limited understanding of phage-lung interactions. This work explores a workflow for administering purified phages to macrophage models to evaluate their potential as therapeutic vectors and macrophage immunomodulating effects. First, crude T4 phage lysates were purified from endotoxins using spin columns (SP) and 1-Octanol organic phase extraction (OP). Endotoxin concentrations were quantified via Limulus Amebocyte Lysate assay, and phage DNA concentrations were measured by absorbance spectroscopy. Both purification methods reduced the endotoxin concentration by 10,000-fold using spin columns and 26-fold using organic solvent extraction. However, the spin column purification resulted in a 3-4 fold reduction in phage DNA and a significant reduction in phage titer. The second aim of this thesis is to compare purified phage preparations and crude phage lysate in a macrophage model to evaluate phage internalization and clearance, effects on mammalian cell metabolic activity, and modulation of macrophage inflammatory responses and antigen presentation. SYBR Gold-stained phages were visualized in RAW 264.7 stained with Hoechst and CellMask Plasma membrane stain, where phages localized in the cytoplasm and surrounded the nucleus of cells. Macrophage metabolic activity post-phage treatment was measured using Cell Titer-Glo. Flow cytometry was conducted to assess macrophage polarization and immune response. RAW 264.7 cells treated with purified lysates demonstrated significantly higher metabolic activity at 24 hours compared to lipopolysaccharide (LPS controls, indicating that purified phages are non-cytotoxic to macrophage function. The crude and purified T4 phages were then co-administered with LPS to mimic an LRI inflammatory environment. RAW 264.7 cells activated with LPS show greater internalization of T4 phages. Macrophages dosed with purified T4 phages expressed minimal CD40+, a pro-inflammatory marker, compared to crude T4 phage lysate and LPS. Maximal phage internalization and CD40+ expression were observed at 24 hours, followed by phage clearance by 72 hours, corresponding with a resolution of inflammation. These findings demonstrate that endotoxin-free phage samples can exhibit dynamic immunomodulatory effects in macrophages. Future work will focus on improving endotoxin purification methods that minimize phage loss. Additionally, complex in-vitro models, including air-liquid interface cultures, diverse phage types and immune cell populations, and bacterial co-culture systems, will be developed to evaluate phage lytic activity within mammalian cells and understand how macrophage clearance may limit therapeutic efficacy in the respiratory system.