Fetal bovine serum (fbs)

NIH:OVCAR-3 cell line was grown in RPMI-1640 culture medium (Sigma-Aldrich, Germany), supplemented with 2 mM L-glutamine, 100 U/mL penicillin/100 µg/mL streptomycin (Sigma-Aldrich), 15% fetal bovine serum (FBS) (Sigma-Aldrich, Germany), and 0.01 mg/mL bovine insulin (Sigma-Aldrich). A2780 and A2780 Cis cell lines were cultivated in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (Sigma Aldrich), 2 mM L-glutamine, 1% antibiotics (penicillin + streptomycin) (all reagents from Sigma Aldrich) and with 1 µM cisplatinum in the case of A2780 Cis cell line. Ishikawa cell line was cultivated in Eagle’s Minimal Essential Medium (MEM) supplemented with 10% FBS, 2 mM L-glutamin, 1 mM sodium pyruvate, 1% NEA, and 1% antibiotics [25 (link)]. BJ HEP cell line was cultivated in Eagle’s Minimal Essential Medium (MEM), supplemented with 10% Fetal Bovine Serum (Sigma Aldrich), 2 mM L-glutamine, 1% antibiotics (penicillin + streptomycin), and 1% Non-Essential Aminoacids (NEA) Solution (Sigma Aldrich) [26 (link)]. HUVEC cell line was cultivated in RPMI medium, with 10% Fetal Bovine Serum (Sigma Aldrich), 2 mM L-glutamine, and 1% antibiotics (penicillin + streptomycin) (all reagents from Sigma Aldrich). Cell cultures in the 12th passage were used [27 (link)].

HEK293T cells were obtained from ATCC, and then maintained with DMEM (Gibco, Gaithersburg, MD) containing 10% FBS (Sigma). The human SCLC cell lines were obtained from ATCC. NCI-H748, NCI-H1963, and NCI-H226 were maintained with ATCC-formulated RPMI-1640 medium containing 10% FBS (Sigma). NCI-H1882 cells were maintained with ATCC-formulated DMEM/F12 cell culture media containing 10% FBS (Sigma). Mouse KP1 and KP3 were maintained with ATCC-formulated RPMI-1640 medium containing 10% FBS (Sigma).

The HeLa cell line was obtained from the American Type Culture Collection (ATCC CCL-2; Manassas, VA, USA) and was maintained in minimum essential medium (MEM; Corning Cellgro; Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich; St. Louis, MO, USA) and 1% penicillin–streptomycin solution (Hyclone; Logan, UT, USA). SK-N-MC cells were obtained from ATCC (HTB-10) and maintained in MEM supplemented with 10% FBS (Sigma Aldrich; St. Louis, MO, USA), 1% penicillin–streptomycin solution (Hyclone; Logan, UT, USA), 1% sodium pyruvate (Hyclone; Logan, UT, USA), and 1% non-essential amino acid solution (Hyclone; Logan, UT, USA). HUH-7 cells were obtained from the JCRB Cell Bank (JCRB0403) and were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning Cellgro; Manassas, VA, USA) supplemented with 5% FBS (Sigma Aldrich; St. Louis, MO, USA) and 1% penicillin–streptomycin solution (Hyclone; Logan, UT, USA). Finally, Vero cells (ATCC CCL-81) were maintained in MEM (Corning Cellgro; Manassas, VA, USA) supplemented with 5% FBS (Sigma Aldrich; St. Louis, MO, USA) and 1% penicillin–streptomycin solution (Hyclone; Logan, UT, USA). Cells were maintained at 37 °C and 5% CO₂. Cells were split twice weekly to maintain subconfluency.

A549 cells (non-small cell lung cancer; CCL-185) were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium (Corning) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). A375 human melanoma cells (CRL-1619) were also obtained from ATCC and cultured in high-glucose DMEM (Gibco) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). MRC-5 human lung fibroblasts (CCL-171) and BJ foreskin fibroblasts (CRL-2522) were also obtained from ATCC and maintained in EMEM (ATCC) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). HEK293T cells were obtained from GenHunter and cultured in high-glucose DMEM (Gibco) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin–streptomycin (Gibco). All cell lines were maintained at 37 °C and 5% CO₂. All cell lines were routinely tested for mycoplasma and were at all times mycoplasma negative.

Pancreatic cancer cell-line PANC-1 was procured from ATCC (CRL-1469; Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (30–2002, ATCC) supplemented with 10% FBS (Sigma, St.Louis, MI, USA). MIA PaCa-2 was obtained from ATCC (CRM-CRL-1420) and cultured in Dulbecco’s Modified Eagle’s Medium (30–2002, ATCC) supplemented with 10% FBS. AsPC-1 was obtained from ATCC (CRL-1682), and cultured in an RPMI-1640 medium (30–2001, ATCC) supplemented with 10% FBS (Sigma). Capan-1 was obtained from ATCC (HTB-79), and cultured in an Iscove’s Modified Dulbecco’s Medium (30–2005, ATCC) supplemented with 20% FBS (Sigma).
A human pancreatic nestin-expressing cell line,^{27 (link)} hTERT-HPNE, was obtained from ATCC and cultured in amixture of 75% DMEM without glucose (Cat#. D-5030; Sigma, with additional 2 mM L-glutamine and 1.5 g/L sodium bicarbonate) and 25% Medium M3 Base (Cat# M300F-500; Incell Corp., San Antonio, TX, USA) supplemented with 10% FBS (Sigma). All the cells were cultured at 37°C in 5% CO₂.

FBS (Sigma-Aldrich, St. Luis, Missouri, USA) was used to prepare protein corona of NPs. FBS was used as supplied stock solution (100% FBS) or diluted ten times (10% FBS) in PBS without CaCl₂ or MgCl₂ (Sigma-Aldrich, St. Luis, Missouri, USA). LoBind micro-centrifuge tubes (Eppendorf, Hamburg, Germany) were used for all experiments. Prior to corona preparation, we dispersed NPs to a final concentration of 1 mg/ml in the four analysed media: PBS (Sigma-Aldrich, St. Luis, Missouri, USA), 0.9% (m/v) NaCl (B Braun, Melsungen, Germany), RPMI (Gibco Laboratories, Gaithersburg, Maryland, USA) or dH₂O. The pH of dispersion media was held constant for all experiments. Properties of dispersion media are reported in Table 1. Dispersions were vortexed and incubated for 5 min at room temperature. Afterwards, 100 μl of NPs dispersion (containing 100 μg of NPs) was added to 1 ml of 100% or 10% FBS, vortexed and incubated for 1h at 37°C. FBS-NPs mixtures were transferred to a new microcentrifuge tube and centrifuged at 15000 × g for 20 min at 4°C. Supernatants were removed and pellets dispersed in 1 ml of cold PBS without CaCl₂ and MgCl₂. This procedure was repeated three times. Proteins that remained adhered to NPs were considered as their protein corona [40 (link)]. Control samples were prepared in identical way with equal volume of dispersion media added instead of NPs.

All cells were purchased from the Taiwan Bioresource Collection and Research Center (Hsinchu, Taiwan). The MDA-MB-231, HCC1937, T-47D and ZR-75-1 were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (Gibco, Grand Island, NY). The BT474 was cultured in 90% Hybri-Care Medium (Sigma-Aldrich, St. Louis, MO) with 30 ng/ml EGF and 10% fetal bovine serum. The MDA-MB-453 was cultured in 90% Leibovitz's L-15 medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum and without CO₂. The MCF-7 and Hs 578T were cultured in Dulbecco's Modified Eagle Medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum. The MDA-MB-361 was cultured in 80% Leibovitz's L-15 medium with 20% fetal bovine serum and without CO₂. The U-2 OS was cultured in McCoy’s 5A medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum.