Manipulating PLGA nanoparticle cellular uptake and trafficking
Nanotechnology is one of the most exciting and fast-moving areas of science today. A wide range of medical applications use nanoscale materials in the diagnosis, treatment, and prevention of various diseases. Nanoparticles (NPs) are being extensively studied as drug delivery systems due to their ability to minimize toxicity and improve efficacy. Poly lactic-glycolic acid is one of the most commonly used synthetic polymers for drug delivery due to its characteristics such as biodegradability and controlled drug release. Nanoparticle properties, such as size, play a key role in their interaction with the cell membrane and can lead to the initiation of specific endocytic mechanisms. Antimicrobial resistance has become a global problem as a result of antibiotic overuse, and misuse. Bacteria have developed multiple strategies to survive antimicrobial therapy, including bacterial pathogens hiding inside cells, such as phagocytes, preventing antibiotic activity. Several extracellular bacterial pathogens including P. aeruginosa, S. aureus, and K. pneumonia can invade, survive, and reproduce inside human cells. Therefore, finding effective treatments to eradicate these intracellular bacterial infections is critical. The aim of this research was to improve the efficacy of drug-loaded PLGA NPs through manipulating their cellular uptake and intracellular trafficking to a specific compartment inside the cell by modulating physicochemical properties i.e. size. Different sizes of Rhodamine-B conjugated PLGA NPs were synthesized and optimized to achieve the required sizes (< 100, ≈ 200, > 500 nm). Through confocal microscopy, NP internalization was explored in five different epithelial (A549, 16HBE14o-, HCT116, HeLa, and Panc1) and two macrophage cell lines (RAW264.7 and THP1). In addition, the dependance of NP uptake upon the clathrin-dependent endocytosis pathway was investigated. In order to translate our observations in regard to NP size-dependent uptake, ciprofloxacin-loaded and ceftazidime-loaded PLGA NPs were formulated and size-dependent antibiotic-loaded PLGA NP activity was tested against three different intracellular bacterial pathogens in a bacterial-macrophages co-culture in vitro model. Cellular uptake studies in immune cells demonstrated a consistent increase in NP uptake with increased particle diameter and NP localization with lysosomal compartments was confirmed. Despite variation in rate and extent, preferential uptake of medium-sized NPs was observed in epithelial cells. However, the cellular internalization of NPs in epithelial cells was inconsistent in its nature, and in its reliance upon clathrin. Significant size-dependent antimicrobial activity of antibiotic-loaded PLGA NPs was demonstrated in K. pneumoniae. However, despite its potent bactericidal activity against S. aureus, free and NP-loaded ciprofloxacin had no activity against intracellular S. aureus probably due to bacterial concealment in vacuoles the NPs did not reach. In our work, advantageous size-dependent targeting to immune cells was proven, yet, full-time dedicated research utilizing a variety of complementary methods is required to draw a clear road map describing NPs cellular internalization, endocytic mechanisms involved and intracellular trafficking. Thesis embargoed until 31 July 2027.
Nanoparticles, PLGA, cellular uptake, endocytosis, ESKAPE, antimicrobial resistance
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