Infinite f200 spectrophotometer

Recombinant His-HAT1 protein or cell lysate (100 ng) diluted in coating buffer (Seracare, Biogen Cientifica, Madrid, Spain) was plated in a microtiter plate of 96 wells (NUNC, ThermoFisher Scientific, Massachusetts, USA) and incubated overnight at 4 °C. The plate was then blocked using BSA 0.2% in selection buffer and incubated for 1 h at room temperature, followed by washing the plate three times in PBS-Tween 0.1%. The aptamers conjugated with biotin and digoxigenin were diluted in selection buffer to prepare various solutions of different concentrations, as indicated in the figure legends, and structured by first heating for 10 min at 95 °C and then incubating for 10 min on ice. Next, 100 µL of the diluted aptamers were added, and the plate was incubated at 37 °C for 1 h. After three washes with PBS-Tween 0.1%, the plate was incubated with 100 μL of a 1/1000 dilution of streptavidin conjugated with horse radish peroxidase (HRP) (Sigma Aldrich, Madrid, Spain) or HRP conjugated anti-digoxigenin antibody (Roche, Madrid, Spain) for 1 h of incubation at room temperature. Finally, the plate was washed as mentioned above and developed using ABTS solution (Roche, Madrid, Spain). The absorbance was measured at 405 nm in an Infinite F200 spectrophotometer (TECAN, Männedorf, Switzerland).

The human colorectal adenocarcinoma cell line HT-29 (ATCC HTB-38) was grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin, and 2 mM l-glutamine in a humidified 5% CO₂ incubator at 37 °C. Periodical subculturing of the cells was done on average every 4 days. For differentiation, the cells were grown on transwells (Corning™ Falcon™ Cell Culture Inserts) at a density of 1 × 10⁵ cells per mL in 24-well plates (area of the transwell = 0.3 cm²) and differentiated with the abovementioned media supplemented with high glucose (10% v/v) for 21 days.^{22 (link)} TEER measurements (see below) were done to observe the barrier integrity. The media was replaced every two days. In our pilot studies, cytotoxicity towards HT-29 cells was determined based on LDH release using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega). To this end, cells were seeded in a 96-well plate at a density of 5 × 10⁵ cells per mL without differentiation (no glucose) for 24 h and then exposed to a range of concentrations (0.1–100 μg mL⁻¹) of COOH-PS NPs and NH₂-PS NPs. After exposure, supernatants were collected and processed for LDH release according to the manufacturer's instructions. The samples were analyzed on a Tecan Infinite® F200 spectrophotometer (Männedorf, Switzerland).

After exposure to NH₂-PS and COOH-PS NPs, cytotoxicity was assessed using the Alamar blue assay (Thermo Fisher Scientific, Sweden), as described.^{25 (link)} Briefly, the cells grown in transwells were first exposed to the Alamar blue dye directly and incubated for 2 h. The media containing the dye was then removed and added to the 96-well plate. The samples were analyzed on a Tecan Infinite® F200 spectrophotometer (Männedorf, Switzerland). Cell viability was quantified by the % cell viability versus the negative control value, which was set as 100%. Lysed cells served as positive control.

Plates were coated with 10 μg/mg type I collagen (Sigma, St. Louis, MO, USA) or BSA (Sigma, St. Louis, MO, USA) as a control for 1 h at 37 °C. Cells were seeded at 5 × 10⁴ cells/well in 6-well plates and transfected with aptamers 16–24 h later. After 24 h, cells were collected, added to coated plates at 3 × 10⁴ cells/well in serum-free medium, and allowed to bind at 37 °C for 1 h. Wells were washed twice with PBS to remove non-adhered cells and an MTT assay was performed with 1 h of incubation between the addition of the MTT reagent and lysis buffer. After 24 h, absorbance was read at 540 nm in an Infinite F200 spectrophotometer (TECAN, Männedorf, Switzerland).

NPs were assessed for lipopolysaccharide (LPS) content using the chromogenic endpoint Limulus Amebocyte Lysate (LAL) assay (Lonza, Walkersville, MD, United States) (Bhattacharya et al., 2017 (link)). The absorbance was measured on a Tecan Infinite^® F200 spectrophotometer (Männedorf, Switzerland). The NPs were all endotoxin-free.

For cytotoxicity assessment, cells were seeded in 96-well plates at a density of 1.5 × 10⁴ cells/cm² 1 day before the experiment. The following day, NP dispersions were prepared in cell culture medium and added to the cells to achieve final concentrations of 0, 0.01, 0.1, 1, 5, 10, 20, and 50 μg/ml. Following 24 or 48 h of exposure, supernatants of exposed cells were collected in a fresh 96-well plate and lactate dehydrogenase (LDH) release was monitored (Mukherjee et al., 2020 (link)) using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay kit (Promega). Samples were analyzed using a Tecan Infinite^® F200 spectrophotometer (Männedorf, Switzerland). Results are expressed as % LDH release versus maximum LDH release (cell lysis). The Alamar blue assay for metabolic capacity was also performed. Cells exposed as above were incubated with the Alamar blue dye (Thermo Fisher Scientific) for 4 h, and fluorescence was analyzed using a Tecan plate reader as described elsewhere (Keshavan et al., 2021 (link)).