Nanocarriers for cell-specific targeting in the lung and development of an in vivo validated in-vitro screening platform of drug uptake and release

Ishaque, Mehwish (2025): Nanocarriers for cell-specific targeting in the lung and development of an in vivo validated in-vitro screening platform of drug uptake and release. Dissertation, LMU München: Medizinische Fakultät

Abstract

Over the past few decades, there has been a remarkable increase in the utiliza-tion of nanomaterials/particles in various industries, including textiles, infor-mation technology, food, electronics, and medical sciences. Nanoparticle-mediated drug delivery is an emerging therapeutic approach for various respira-tory diseases. Several forms of nano-carriers, such as polymeric, liposomes, carbon, gold, micelles, and biological particles, are used for targeted drug deliv-ery. The nano-carrier must be stable, biocompatible, and biodegradable for successful drug delivery to targeted cells. The alveolar epithelium has a large surface area and is a significant drug interaction and absorption target. Nanocarrier-mediated drug delivery has the advantage of delivering a high con-centration of drugs to the targeted cells, such as alveolar epithelial cell types (AT I and AT II) and alveolar macrophages. Most studies have investigated the cellular interaction of particles through ani-mal models, which can be expensive and time-consuming. To avoid animal models, in vitro exposure methods provide an efficient way to study these inter-actions and their effects on the cellular level. This study used conventional submerged and air-liquid interface (ALI) exposure systems to observe the tar-geted delivery of particles to the alveolar cells. The thesis is divided into two studies with different objectives. The first section of this study aimed to investigate the cell-specific uptake of different-sized and anionic or cationic fluorescent-labeled polystyrene latex par-ticles (PS) as carrier surrogates and their toxicological side effects in different in vitro exposure systems. The study used alveolar epithelial cells and macro-phages in conventional submerged and air-liquid interface (ALI) culture setups. Submerged exposures are more accessible to perform, but ALI exposures are more physiologically realistic and potentially more biologically meaningful. Amine-surface modified (PS NH2) (0.1 and 1 µm), carboxyl-surface modified (PS COOH) (0.03, 0.5, and 1 µm), and sulfate-surface modified (PS SO4) (0.1 and 0.5 nm) polystyrene latex beads (PS) were studied. The LA-4 (epithelial, type II-like mouse lung adenoma cells) and MH-S (immortalized mouse alveolar macrophages) murine cell lines were exposed to polystyrene latex beads for 24 hours at 37°C in both ALI and submerged conditions, and the uptake was quan-tified using mean fluorescence intensity per cell assessed by flow cytometry. Cells were also evaluated for cell viability (WST assay) and cytotoxicity (LDH assay) after 24 hours of exposure. The study found that in submerged conditions, epithelial cells most effectively took up larger COOH-PS and NH2-PS particles (1 µm). At the same time, mac-rophages internalized 1 µm carboxyl-PS particles the best, and sulfate-surface modified PS particles showed a minor uptake for all cell types. However, re-gardless of surface modifications, all cell lines showed stronger uptake signals at the air-liquid interface, especially for 1 µm particles. The study also found that toxicity results differed between submerged and air-liquid interface culture conditions, with more toxicity observed in air-liquid interface conditions for all particles. The amine-surface modification, especially at 0.1 µm, was identified as the most toxic modification for all cell types, particularly for macrophages. These results suggest cell-targeting studies using nano-carriers should be con-ducted in an air-liquid interface miming the respiratory surface. To determine their cell-specific uptake, the study's second section investigated the uptake of differently sized fluorescently labeled biodegradable PLGA parti-cles in various models (in vitro and in vivo). I chose to use PLGA particles in-stead of PS particles because PS particles cannot provide controlled release of encapsulated drugs or exhibit biocompatible properties and are non-biodegradable, non-bioabsorbable, and non-biocompatible. Therefore, their use is limited in biomedical applications that involve interaction with biological sys-tems. However, PS particles are widely used in basic research due to their low cost, ease of preparation, and uniform size. Nevertheless, for biomedical pur-poses, alternative materials like biodegradable polymers (e.g., PLGA) or natural polymers (e.g., chitosan, alginate) are more appropriate because they provide excellent biocompatibility and controlled drug release capabilities. The study exposed monocultures and co-cultures of alveolar epithelial and alveolar mac-rophage cell lines to different-sized PLGA particles under conventional sub-merged or air-liquid interface conditions and compared the uptake efficacy with that observed in the lungs of mice. The study investigated 0.1 µm, 0.5 µm, and 1 µm PLGA particles. I studied the effects of PLGA particles on mouse lung cells in vitro and in vivo. I used LA-4 and MH-S cell lines in monocultures and co-cultures and exposed them to PLGA particles for 24 hours under submerged and ALI conditions. I also exposed C57BL/6J mice to PLGA particles (sizes: 0.1 and 1 µm) via intratracheal instillation and analyzed them after 24 hours. We used confocal microscopy and flow cytometry to monitor and quantify particle uptake. Our results showed that particle uptake varied based on particle size and cell type and was influenced by culture conditions. In a submerged state, epithelial cells exhibited the highest uptake of the largest particles (1.0 µm), whereas macrophages were equally effective at taking up particles of 0.5 µm and 1.0 µm. However, under ALI conditions, there was a significantly high up-take of all particle sizes (0.1 µm, 0.5 µm, and 1 µm) by LA-4 cells in both mon-oculture and co-culture scenarios and also a significantly higher uptake of smaller particles of 0.1 and, 0.5 µm size as compared to 1 µm particles by MH-S cells. When 0.1 and 1 µm particles were delivered to mouse lungs, AT2 cells showed the most significant uptake for 0.1 µm particles, followed by 1 µm parti-cles. In comparison, alveolar macrophages showed the most considerable up-take of 1 µm particles, followed by 0.1 µm particles. The absence of toxicity of these particles was observed in both in vitro and in vivo experimental settings. In conclusion, our study suggests that the correlation between particle uptake in the ALI co-culture system and in vivo epithelial cells varies depending on the cell type and particle size. While the results were similar for smaller particles, a different pattern of particle uptake was observed in BAL macrophages and AT2 cells for larger particles. Therefore, more research is needed to understand the underlying factors contributing to these discrepancies, including cell type and dose, to improve the accuracy of in vitro models for predicting in vivo particle uptake.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 8. Mai 2025
• 1. Bericht­erstatter:in: Meiners, Silke