*2.8. In Vitro Cytotoxicity/Anti-TNFα Efficacy*

Cell culture: The HEK001 cell line was obtained from ATCC (Promochem Partnership, Manassas, VA, USA). HEK001 was grown in monolayer on a solid support, at 37 ◦C in a humidified atmosphere with 5% CO<sup>2</sup> and maintained in keratinocyte serum-free media supplemented with 100 µg/mL epidermal growth factor (EGF) (Life Technologies, Carlsbad, CA, USA), 20 U/mL penicillin, and 20 µg/mL streptomycin (Life Technologies). Mycoplasma-free maintenance was confirmed every two weeks by PCR amplification.

Cell treatments: For the cell viability assays, HEK001 cells were seeded at a density of 5000 cells/well in 96-well culture plates. At 24 h post-seeding, cells were treated with increasing doses of DEX-charged LPNCs, non-charged LPNCs, or the corresponding amount of benzalkonium chloride for 24 h. After 48 h, cell viability was assessed using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) colorimetric assay (Sigma-Aldrich, St. Louis, MO, USA). Cell survival for all experiments was expressed as the percentage of viable cells, relative to that in untreated cells (defined as 100%). For the cytokine modulation assays, HEK001 cells were seeded at a density of 700,000 cells/well in 6-well culture plates. At 24 h post-seeding, cells were pre-treated with 10 µg/mL of lipopolysaccharide (LPS) for 1 h and then treated with 0.1 mM of DEX or DEX-loaded LPNCs for 24 h.

RNA isolation and RT-PCR: Total RNA was isolated from cultured cells using the SV Total RNA Isolation System (Promega, Madison, WI, USA), following the manufacturer's instructions. A total of 1 µg of RNA was reverse transcribed to cDNA with M-MLV Reverse Transcriptase (Life Technologies) and random hexamers (Life Technologies). Analyses of the TNFα and GAPDH (endogenous control) mRNA levels were performed by RT-PCR, using commercial TaqMan gene expression assays (Applied Biosystems). Relative quantification of gene expression was assessed using the ∆∆CT method, as described in the TaqMan user manual (User Bulletin no. 2; Applied Biosystems). Gene expression levels

for TNFα were normalized to the GAPDH gene. The amounts of mRNA are expressed as arbitrary units.

Statistical analysis: Results from the cell viability and cytokine modulation were statistically analyzed by using GraphPad Prism (8.0.2, 2019, La Jolla, San Diego, CA, USA), with Student's paired *t* test for cytotoxicity assays and two-way ANOVA for cytokine modulation assays. In all cases, differences were considered significant when the *p*-value < 0.05.
