Polyethylene glycol

EVs were harvested from the cells 48 h after transfection with LMP1. The collected EVs were enriched using the ExtraPEG method, as previously described (74 (link)). Briefly, the medium was centrifuged at 500 × g for 5 min and at 2,000 × g for 10 min in an Eppendorf 5804R centrifuge using an S-4-104 rotor, followed by 10,000 × g for 30 min in an Eppendorf 5804R centrifuge using an FA-45-630 rotor to remove cells and cellular debris. Subsequently, a 1:1 volume of 16% (2×) polyethylene glycol (average M_n, 6,000) (catalog number 25322-68-3; Alfa Aesar) and 1 M sodium chloride was added to the samples, and the samples were incubated overnight at 4°C. The incubated samples were centrifuged at 3,214 × g for 1 h in an S-4-104 rotor. The pellet was then washed with 1× phosphate-buffered saline (PBS) and centrifuged at 100,000 × g for 70 min in a Beckman Max-E centrifuge using a TLA120.2 rotor. The collected EV samples were resuspended in particle-free PBS for nitrilotriacetic acid (NTA) or resuspended in 2× Laemmli sample buffer (4% SDS, 100 mM Tris [pH 6.8], 0.4 mg/ml bromophenol blue, 0.2 M dithiothreitol [DTT], 20% glycerol, 2% β-mercaptoethanol [BME]) for immunoblot analysis.

Curcumin was supplied from TCI (Tokyo Chemical Industry Co., Ltd. Tokyo, Japan). Hydroxy propyl methylcellulose (HPMC), carboxy methylcellulose sodium (Na CMC), glycerol (GLY), polyethylene glycol (PEG 400), triethylacetate 99% (TEC) were supplied by Alfa Aesar, Heysham, England. Polyvinyl alcohol (PVA) molecular weight (MW) 31kDa–50 kDa, 98–99% hydrolysed, Pluronic F127 and cellulose membrane MW cut-off 12 kDa–14 kDa were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Large-diameter SWCNTs (Tuball™, batch: 89-21082015, OCSiAl, Luxemburg), Dextran (DEX, average molecular weight of 70,000 Da, PanRecAppliChem, Germany), poly(ethylene glycol) (average molecular weight of 6000 Da, Alfa Aesar, Germany), sodium cholate (SC, PanRecAppliChem, Germany), sodium dodecyl sulfate (SDS, Sigma-Aldrich, St. Louis, MO, USA), methanol (Avantor, Poland), H₂O₂ (hydrogen peroxide, Sigma-Aldrich, USA) were all purchased from commercial sources. The chemical compounds had a p.a. class. The Millipore Elix 10 demineralization system (Milipore SAS, Molsheim, France) was used to prepare demineralized water for the study. The quality of the water was monitored by measuring its electrical conductivity.

Size-exclusion chromatography experiments were performed on an Agilent 1100 series HPLC value system equipped with a 96-well autosampler and UV–visible detector. Wild-type and mutant Smlt2602 at a final concentration of 10 μg/mL was added to poly-ManA or poly-GlcA at a final concentration of 1 mg/mL in 20 mM sodium phosphate buffer, pH 8.5, and incubated for 6 h at room temperature. The reaction mixture (15 μL) was injected into a TSKgel SuperOligoPW column (Tosoh) equipped with corresponding guard column. The mobile phase was 20 mM sodium phosphate buffer (pH 8.5) plus 250 mM NaCl and the flow rate was 0.3 mL/min. Unsaturated uronic acid products were detected by absorbance at 235 nm. Polyethylene glycol (molecular weight 600, 1000 and 2000; Alfa Aesar), d-glucuronic acid (Acros Organics) and xylose (Spectrum) were used as molecular weight standards to generate a calibration curve.