Influence of Hydrophobicity on Nanoparticle-Induced Lung Injury   Anna Babin Morgana,b, Domenico Spinaa,b, Yanira Riffo-Vasqueza,b, Aateka Patela,b, Hanpeng Chena, Anna Starra, Stuart Jonesa, Ben Forbesa, Kavitha Sunasseec, Stephen Clarkc, Rafael Torres Martin De Rosalesc, Clive Pagea,b, and Lea Ann Daileya aInstitute of Pharmaceutical Science, King’s College London, 150 Stamford Street, SE1 9NH London, UK bSackler Institute of Pulmonary Pharmacology, King’s College London, 150 Stamford Street, London SE1 9NH London, UK cDivision of Imaging Sciences & Biomedical Engineering, King’s College London, 4th Floor Lambeth Wing, St Thomas Hospital, London SE1 7EH, UK RATIONALE. Nanomaterials as drug vehicles have the potential to improve inhaled medicines, but must be shown safe for the lungs. To investigate the influence of nanomaterial hydrophobicity on lung injury, this longitudinal study quantified markers of lung injury in mice, following pulmonary exposure to sterically stabilised low or high hydrophobicity poly(vinyl acetate-co-alcohol) nanoparticles (~150 nm), with a view to better understanding pulmonary nanotoxicity. It was hypothesized that increased hydrophobicity would be linked to increased toxicity over time.   METHODS. Relatively low or high hydrophobicity nano-suspensions were produced from poly(vinyl acetate-co-alcohol), by varying the polymer’s acetate content proportional to OH content through differential hydrolysis, as confirmed by nuclear magnetic resonance (NMR). The resultant low or high hydrophobicity polymeric suspensions were labelled PVAc70% (30% OH) and PVAc85% (15% OH), respectively. Hydrodynamic particle size was confirmed using dynamic light scattering (DLS: Malvern Zetasizer Nano). In addition, ultrafine silica (Min-U-Sil 5® quartz dust) was investigated as a particle control, and bleomycin A5 HCl was investigated as a fibrotic-associated control for lung injury. Data were calculated as mean ±sd, using a student t-test for comparison. For dosing to the lungs, male mice (C57BL/6j 22-25 g) received oropharyngeal aspiration (OA) of PVAc nanoparticles (300 µg), silica (300 µg), bleomycin (0.05 mg/kg body weight, 6x uid), or respective vehicles (40 µL). For conventional toxicological comparisons, animals were sacrificed at 1, 7, and 28 days post-treatment. Data were analysed using ANOVA with Bonferroni test. For non-invasive assessment, micro-computed tomography (CT) captured baseline images for all mice pre-treatment. Mice then received PVAc nanoparticles (or vehicle), silica, or bleomycin, followed by CT assessment at 1, 7, and 28 days post-treatment. Results were compared to naïve and baseline images, allowing each animal to be used as its own control (ANOVA-RM with Dunnett test). RESULTS. NMR confirmed significant polymer hydroxylation for PVAc70% (31.9 ±1.4% OH, n=7), versus PVAc85% (16.2 ±1.7, n=5, p<0.001). DLS confirmed that particle sizes for PVAc70% and PVAc85% suspensions did not differ significantly (168 ±12 and 143 ±4 nm, n=8). Observed difference in nanotoxicity between PVAc suspensions were thus attributed to relative hydrophobicity, not differences in particle size.   Conventional assessments included bronchoalveolar lavage (BAL) analyses and histological quantification of picrosirius factor (PF) for fibrosis. For BAL assessment, lung injury in PVAc, silica, and bleomycin groups was detected by elevated cytokines, neutrophils, or total protein, which was not detected in vehicle groups. In comparison to PVAc70%, treatment with PVAc85% was generally associated with increased BAL parameters, peaking at day 1 and resolving by day 7. For PF histological assessment, area of picrosirius red-stained collagen from 3 lung slices/animal (~4 µm: upper, middle, and lower left lobe) was expressed as percentage total coverage. Silica and PVAc85% demonstrated significantly (p<0.05) elevated PF scores at day 7 only. In contrast bleomycin showed increasing collagen deposition over time (p<0.05 day 28). CT assessment detected significant increases in high-density signal volumes in bleomycin-treated animals at all times post-treatment (p<0.001). PVAc85% demonstrated a significant increase at day 7 only (p<0.05). Increased signals in experimental groups peaked at day 7, and were not directly related to BAL changes or collagen deposition. CONCLUSIONS. Increased hydrophobicity of polymeric nanoparticles was associated with an acute inflammatory response, which resolved within 7 days of administration. Although longitudinal CT analysis of high-density signals did detect comprehensive changes in lung tissue over time, it lacked the specificity to discriminate between purely cellular inflammation or fibrotic changes. Relative hydrophobicity of polymeric PVAc nano-suspensions was predicted to be linked to increased lung inflammation. Mice received graded hydrophobic nano-suspensions, control treatments, or vehicle(s). BAL inflammatory parameters (a-f) were measured using standard H&E, ELISAs, or Bradford protein assay (mean ±sd). Notably, PVAc85% induced a prominent influx of polymorphonuclear cells (PMNs), compared to PVAc70%. In contrast, PVAc70% demonstrated a high number of vacuolized mononuclear cells (MNCs), the role of which in inflammation is not entirely understood. As predicted, PVAc85% resulted in greater collagen deposition and elevated CT signals compared to PVAc70% (mean ±sem, n=~3-4, CI 95%) (*p<0.05, ***p<0.001). Neither of the PVAc nano-suspensions resulted in sustained significant changes. This data confirmed that relative hydrophobicity was linked to increases in typical parameters of lung inflammation and injury. However, the role of vacuolized cells in lung health following application of nanomaterials remains to be investigated.