Acetone

Native hyaluronan of three different molecular weights (106 kDa, 420 kDa and 1.46 MDa) was obtained from Contipro (Czech Republic). The molecular weights were determined by the producer using the SEC-MALLS method. Stock solutions of hyaluronan were prepared in deionized water to a concentration of 0.5 g∙L⁻¹.
Pyrene, prodan, and perylene of fluorescence grade were purchased from Fluka (Czech Republic). Stock solutions of fluorescence probes were prepared in acetone p.a. obtained from LachNer (Czech Republic). The concentrations of stock solutions were 2∙10⁻⁴ mol∙L⁻¹ in the case of pyrene and prodan, and 1∙10⁻⁴ mol∙L⁻¹ in the case of perylene.
Tert-butanol (anhydrous ≥ 99.5%) was obtained from LachNer (Czech Republic).
For the preparation of samples, fluorescence probes in acetone were added to freeze-drying flasks and the acetone was evaporated. Subsequently, 10 ml of TBA was introduced and the samples were mixed on a vibration shaker until probe dissolution. Then, 40 ml of hyaluronan stock solution was added. The samples were stirred constantly until their homogenization. The prepared samples were frozen at −105°C for 12 hours.
Solutions pH was checked and no significant difference between the mixture prepared for freeze-drying and corresponding hyaluronan solution was found; pH was around 5.6.

Styrene, maleic anhydride (MA), p-divinylbenzene (DVB) and 2,2’-azo-bis(isobutyronitrile) (AIBN) were purchased from Sigma-Aldrich (Prague, Czech Republic). Styrene was distilled, MA was resublimed and AIBN was recrystallized from methanol prior to use. Ethyl acetate, ethanol and acetone of analytical grade were obtained from Lach-Ner (Neratovice, Czech Republic). Acetonitrile (ACN) and water of HPLC grade were purchased from VWR (Prague, Czech Republic). Phosphate buffered saline (PBS) solution (pH 7.4) was prepared from tablets supplied by Sigma-Aldrich (Prague, Czech Republic) according to the manufacturer’s instructions. Fetal bovine serum (FBS) was purchased from Sigma-Aldrich (Prague, Czech Republic). Cell culture medium (CellGRO) and AlamarBlue Assay were purchased from ThermoFisher Scientific (Prague, Czech Republic), 24-well plates were from TPP (Prague, Czech Republic).

The structure-directing agent (SDA) for IWW synthesis is 1,5-Bis-(methylpyrrolidinium)pentane dihydroxyde (MPPH), which was prepared according to^{22 (link)}. In total, 40 g of N-methylpyrrolidine (Sigma Aldrich, 97%) was mixed with 37.5 g of 1,5-dibromopentane (Sigma Aldrich, 97%) in 300 ml of acetone (Lachner, 99.99%) and heated under reflux for 24 h. The resulting 1,5-bis-(methylpyrrolidinium)pentane dibromide was then ion-exchanged into th ehydroxide form using Ambersep® 900(OH) an anion exchange resin (Acros Organics, 0.8 mmol of SDA per 1 g of anion exchange resin). The solution was concentrated under low pressure (35 Torr) at 303 K until the hydroxide concentration was ~1.0 M.