4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes

HaCaT cells (CLS Cell Lines Service) were grown in Dulbecco's Modified Eagle's medium (DMEM) cell culture medium supplemented with 4.5 g/L glucose (Merck KGaA, Darmstadt, Germany), 15 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Merck KGaA, Darmstadt, Germany), 2 mmol/L L-glutamine (Merck KGaA, Darmstadt, Germany), 10% FBS Merck KGaA (Darmstadt, Germany), and 1% antibiotic-antimycotic cocktail (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultured at 37°C in a humidified 5% CO₂ environment. Cell culture medium was freshly changed every 2 days. Cells were subcultured every 4 days. Briefly, cells were detached using freshly prepared trypsin/EDTA solution (Merck KGaA, Darmstadt, Germany) and incubated at 37°C for 10 minutes. Cells were collected by centrifugation at 300 x g for 5 minutes. Finally, cells were seeded at a split ratio of 1 : 10. To perform the experiments, harvested and centrifuged cells were counted using a Neubauer chamber and seeded in cell culture plates at a density of 2.5 × 10⁴ cells/mL. Cells were always allowed to attach for 48 hours before starting the experiment. Cells were routinely tested for mycoplasma contamination.

All chemicals were of analytical grade, if not otherwise stated. Sodium chloride (NaCl), sodium hydroxide (NaOH), SDS, 2‐(N‐moprholino)ethanesulfate acid (MES), Tween‐20, sulfuric acid (95–97%, H₂SO₄), uranyl acetate were purchased from Merck (Darmstadt, Germany).
4‐(2‐Hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) (≥99.5%), 2‐propanol, bovine serum albumin (BSA) (≥99.5%), 1,4‐dithiotreitol (DTT), anti‐mouse IgG (γ‐chain specific)‐alkaline phosphatase antibody (#3438), BCIP^®/NBT solution, triton X‐100, glutaraldehyde solution (grade I), acetonitrile (MS grade), formic acid (98–100%), and iodacetamide (≥99%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti‐rabbit IgG (H+L) secondary antibody (#31460), anti‐mouse IgG (H+L) superclonal secondary antibody (#A28177) were purchased from Thermo Fisher (Waltham, MA, USA). SeeBlue^® plus 2 prestained protein standard and 4× LDS sample buffer were purchased from Invitrogen (Carlsbad, CA, USA). C‐LEcta Denarase^® was purchased from VWR (Radnor, PA, USA), HIV‐1 p24 antibody (ab9071) and ACV5 (ab49581) from Abcam (Cambridge, UK), influenza A virus H1N1 HA (GTX127357) from GeneTex (Irvine, CA, USA) and trypsin from Promega (Madison, WI, USA).

Three types of NPs (60 nm diameter) were
purchased from Magsphere Inc. (Pasadena, CA) and included: nonfunctionalized
polystyrene (PS) (plain NP according to manufacturer’s nomenclature),
aminated polystyrene (PS-NH₂), and carboxylated polystyrene
(PS-COOH). Lipids dissolved in chloroform, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from
Avanti Polar Lipids (Alabaster, AL). Sodium chloride and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) were obtained from Merck KGaA (Darmstadt, Germany). All
solutions were prepared using ultrapure water (UW) (Simplicity UV,
Merck KGaA, Darmstadt, Germany) with a resistivity of 18.2 MΩ/cm
at 25 °C and filtered through a 0.20 μm pore size filter
(Scharlau, Spain).

2′,7′-Dichlorofluorescein diacetate (DCFH-DA), trichloroacetic acid (TCA), 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), sucrose, perchloric acid, D-mannitol, imatinib mesylate, Rhodamine123 (Rh 123), 3-(N-morpholino) propane sulfonic acid (MOPS), 2, 4-dinitrofluorobenzene (DNFB), sodium acetate, acetic acid glacial, 2, 4, 6-tripyridyl-s-triazine (TPTZ), iodoacetic acid, dithiothreitol (DTT), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and thiobarbituric acid (TBA) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). Kits for evaluating serum biomarkers of renal injury were obtained from Pars Azmun^® (Tehran, Iran). Ethylenediaminetetraacetic acid (EDTA), reduced glutathione (GSH), malondialdehyde (MDA), fatty acid-free bovine serum albumin (BSA) fraction V, oxidized glutathione (GSSG), ferric chloride hexahydrate (FeCl₃.6H₂O), coomassie brilliant blue, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), meta-phosphoric acid, n-butanol, and 2-Amino-2-hydroxymethyl-propane-1,3-diol-hydrochloride (Tris-HCl) were obtained from Merck (Merck KGaA, Darmstadt, Germany). All salts used for preparing buffer solutions were of the analytical grade and obtained from Merck (Merck KGaA, Darmstadt, Germany).

Poly-(styrene-co-maleic anhydride), cumene terminated (average MW ~1600), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDAC), Hank’s balanced salt solution, advanced Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), bovine serum albumin (BSA), and TrypLE Express were purchased from ThermoFisher Scientific (Dubai, United Arab Emirates). Human recombinant insulin (91077C) was purchased from Merck (Hertfordshire, UK). L-glutamine, penicillin/streptomycin, glucose uptake fluorometric assay Kit, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and pepsin were purchased from Merck (Hertfordshire, UK). All consumables such as petri dishes, plastic tubes (15 mL and 50 mL), cell culture flasks (25 cm² and 75 cm²), transwell plates (CLS3460), and simulated gastric fluids (cat no. 1651) were purchased from Merck (Hertfordshire, UK).

1,5-Carboxy-seminaphtorhodafluor
acetoxymethylester (SNARF-1-AM) and Dulbecco’s modified Eagle’s
medium GlutaMAX (DMEM) were purchased from Thermo Fisher Scientific.
Fetal bovine serum (FBS Supreme), trypsin/ethylenediaminetetraacetic
acid, and penicillin–streptomycin were obtained from PAN-Biotech
GmbH (Aidenbach, Germany). Phosphate buffered saline was bought from
Biozym Scientific GmbH (Hessisch Oldendorf, Germany). Dimethyl sulfoxide
(DMSO) was purchased from Carl Roth (Karlsruhe, Germany). CaCl₂, casein from bovine milk, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), d-glucose, formic acid, HCl, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), KCl, KH₂PO₄, KOH, MeCN, MgCl₂(H₂O)₆, MgSO₄, NaCl, NaHCO₃, (NH₄)₂CO₃, and pepsin from
porcine gastric mucosa were ordered from Merck KGaA (Darmstadt, Germany).
Custom peptides (PVVVPPFLQPEVM, VAPFPEVF, YFYPEL, YQEPVLGPVRGPFPIIV,
and YYVPLGTQ) were synthesized by Genscript Biotech with a purity
of >95% (New Jersey, USA). Double-distilled water (ddH₂O) from Elga Purelab Classic (Veolia Water Solutions & Technologies,
France) was used for all experiments. Krebs-Ringer–HEPES buffer
(KRHB) contains 130 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl₂,
1.2 mM MgSO₄, 1.2 mM KH₂PO_4, 11.7
mM d-glucose, and 10 mM HEPES; the pH was adjusted to 7.4
with KOH.