title: Toxicology and Cellular Interactions of Polymer-based Nanocarriers for Pulmonary Drug Delivery
Creators: name: Mostafa Mahmoud Osman, N
Publisher: name: Liverpool John Moores University
Year: 2023

description: The attractiveness and enhanced applications of nanoparticles (NPs) stem from their exceptional properties at the nanoscale size, i.e., 1-1000 nm, they may exhibit nano-specific toxicological concerns. Hence, the toxicological assessment of NPs and their interactions within biological systems are investigated and continuously evolving to ensure their safety at the nanoscale. This project explored the nanotoxicology potential of NPs fabricated from two polymers; PGA-co-PDL(PG), and PLGA (PL). NPs of different sizes and charges were successfully formulated; 200 nm (PL and PG; to explore chemistry effect), 500 nm and 800 nm (PG only; to explore size effect), and with different charges; negatively- charged using PVA as emulsifier, and positively charged NPs using DOTAP as emulsifier (to explore surface charge effect) via emulsification-solvent evaporation methods. A stability and degradation studies were evaluated in different biological media. All NPs showed better stability in SFM than CM. PL NPs showed faster degradation with acidic pH changes, while PG NPs showed slower degradation with an alkaline pH slowly decreasing toward the neutrality by the end of 28 days. This denoted better suitability of PG NPs for lung delivery with lower acidic changes. A nanotoxicological screen evaluation by a variety of in vitro assays (viability by alamar blue assay, reactive oxygen species by H2DCFDA reagent and Deep Red ROS assay, mitochondrial membrane potential (ΔΨm) by JC-1 dye, cell membrane integrity by LDH Total and Release, cell death; apoptosis and necrosis, caspases activation, inflammatory potentials (IL-8, IL-2, IL-4, IL-6, IL-10, IL-17a, TNF-α, INF-ɣ), and genotoxicity potential by COMET alkaline gel electrophoresis assay) of all NPs were investigated. The NPs showed cytotoxicity that was dependent on the physicochemical nature of the NPs where PG showed biocompatible cellular responses that can be compared to PL at low concentrations as similar or better. The size increase was associated with a limited uptake for the larger sized NPs resulting in lower cytotoxicity at low concentrations to smaller NPs, however, higher cytotoxicity was observed at high concentrations. In addition, negatively charged NPs were reported to be more cytotoxic than their positive counterparts that were correlated to the larger size of positive NPs after dispersion in media that was associated with limited uptake. The underlaying cytotoxicity mechanisms after these NPs exposure demonstrated involvement of ROS, lowering ΔΨm, release or decrease of LDH, apoptosis, caspases activation (Caspase 8 &gt; caspases 3/7 &gt; caspase 9), inflammatory potential (IL-8, IL-6, TNF-α, INF-ɣ), but no genotoxicity was detected. However, there were many challenges on the application of different assays that required optimization to improve the accuracy of the results and ensure the results measured were due to NPs interactions with the cells (to exclude NP interferences). These were addressed and validated. In addition, the integrity of the epithelial barrier was investigated in vitro using Calu-3 polarized tight monolayers grown under ALI to mimic in vivo epithelial lung exposure. The internalisation and uptake mechanisms were investigated under 4 oC, and 37 oC temperatures with the use of a group of pharmacological inhibitors to cover a wide range of endocytic mechanisms after 1 hr exposure to NPs. A visual confirmation and subcellular trafficking for NPs were performed using confocal microscopy. All NPs didn’t show any impairment of tight junctions (TJs). All NPs sowed active endocytic uptake via caveolin, clathrin and macropinocytosis. Visual confirmation of internalisation and co-localisation with lysosomes and mitochondria that confirmed the vesicular transport and possible therapeutic potentials to target subcellular targets. This indicated the potential therapeutic targeting of these NPs to subcellular targets such as lysosomes and mitochondria. Overall, these studies had explored the potential safe use of these polymeric NPs for lung delivery. PG showed better profile of slower degradation (can be used for sustained formulations), of less acidic changes (less risk of acidity and inflammatory stimulation), and biocompatible profile that can be explored for in vivo lung delivery. The different sizes of NPs have shown potential use for lung delivery where the small sized NPs can be used for targeting deep lung while the larger size can be aimed for vaccine targeting to allow macrophages uptake.