Dmem with glutamax

The DMEM with GlutaMax™, trypsin-EDTA, penicillin, streptomycin, FBS Gold were provided by Invitrogen (San Diego, CA, USA) and Annexin V Apoptosis Detection Kit I by BD Pharmingen™ (San Jose, CA, USA). The DMSO, acridine orange, bromide ethidium and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide by Sigma (St Louis, MO, USA). Triazine mustards 12a-d were obtained according to standard procedure described earlier [31 (link)].

HEK293 human embryonic kidney cell line, A549 human cancer alveolar basal epithelial cells and RAW264.7 mouse macrophages (all from ATCC) were used. HEK293, A549 cells were cultured in DMEM with glutamax (Invitrogen) supplemented with 10% FBS (Invitrogen). RAW264.7 cells were cultured in RPMI 1640 with glutamax supplemented with 10% FBS. Cells were grown in tissue culture flasks at 37°C and 5% CO₂.

Cultured U2OS, HT1080, SH‐SY5Y, HeLa, or CV‐1 cells were grown in DMEM with glutamax and 4.5% (w/v) glucose (Invitrogen, Carlsbad, CA, USA) supplemented with 10,000 U/ml penicillin, 10,000 μg/ml streptomycin (Merck Millipore, Darmstadt, Germany), 100 nM Na pyruvate (Sigma‐Aldrich, St. Louis, MO, USA), and 10% (v/v) FCS (Merck Millipore, Darmstadt, Germany) at 37°C and 5% CO₂.
For Drp1 knockdown, the Drp1‐shRNA‐expressing plasmid pREP4 (Lee et al, 2004) was used. Plasmids were transiently transfected by electroporation using the nucleofector Kit V (Lonza, Basel, Switzerland). To enrich transfected cells, selection was performed 24 h after transfection using 200 μM hygromycin B (Life Technologies, Carlsbad, USA) for 2 days, followed by 5–6 days of incubation with lower amounts of hygromycin B (50 μM) in the growth medium.

The mouse endothelial (SVEC4-10) cell line was obtained from American Type Culture Collection (LGC Standards GmbH, Germany). Cells were grown in Dulbecco´s modified eagle´s medium (DMEM with Glutamax; Invitrogen, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS, Invitrogen, Germany). The immortalized human micro vascular endothelial cells (HMEC-1; Centers for Disease Control and Prevention, USA) were cultured in Gibco^® MCDB 131 medium supplemented with 10% (v/v) FCS, 1% (v/v) GlutaMAX^TM I (100×; Life Technologies GmbH, Germany), 1 µg/mL hydrocholesterol (Sigma-Aldrich Chemie GmbH, Germany), and 10 ng/mL epidermal growth factor (Life Technologies GmbH, Germany). Mouse macrophages (J774A.1) used as control cell line were obtained from American Type Culture Collection (LGC Standards GmbH, Germany) and cultured under the same conditions as described above for the SVEC4-10 cells. All used cell lines were cultured at 37 °C in a 5% CO₂ humidified environment. For experimentation, all cells were plated onto a plastic matrix at a density of 1.2 × 10⁴ cells/cm² (endothelial cells HMEC-1), 4.4 × 10³ cells/cm² (endothelial cells SVEC4-10) or 8.8 × 10³ cells/cm² (macrophages J774A.1). They were allowed to grow for 24 h before nanoparticle exposure. The cells were negative for mycoplasma as routinely determined via PCR.

Prior to cell culturing, the NFs and PPF-NFs samples were first sterilized by exposure to UV light for 30 min. Afterwards, HFF cells were seeded onto the samples in a 24-well plate at a density of 40,000 cells/mL. Subsequently, 1 mL of the cell suspension media was added to each well plate. Cell culturing was performed using Dulbecco’s modified Eagle’s medium (DMEM) with glutamax (Gibco Invitrogen, Grand Island, NY, USA) supplemented with 15% fetal calf serum (Gibco Invitrogen, Grand Island, NY, USA), 2 mM L-glutamine (Sigma-Aldrich, Taufkirchen, Germany), 10 U/mL penicillin, 10 mg/mL streptomycin and 100 mM sodium-pyruvate (all from Gibco Invitrogen, Grand Island, NY, USA). The cultures were subsequently incubated at 37 °C under 5% CO₂ for 1 and 7 days (time required for HFFs to adhere and proliferate on the nanofibrous surfaces, respectively). At these time points, cell adhesion, proliferation and viability were evaluated using tissue culture polystyrene (TCPS) as positive control, as will be explained in the following sections.

Human embryonic kidney 293T cells (American Type Culture Collection) and MEFs30 (link)54 (link) were cultured using Dulbecco's Modified Eagles Medium (DMEM) with Glutamax (GIBCO), supplemented with 10% fetal bovine serum (Invitrogen) and antibiotics. Primary mouse hepatocytes were cultured in DMEM—low glucose (GIBCO), supplemented with 5% fetal calf serum (FCS) or normal calf lipoprotein-poor serum (NCLPPS) and antibiotics.