Costar 3513

Two surfaces of the same material (PEDOT:Cl, PEDOT:Heparin, PEDOT:DBS or polyester) were cut (3 × 2.4 cm) with a centrally located tab (1 × 0.5 cm) defining the upper end of the surface. The pair of surfaces was glued opposite to each other in a well of a 12-well plate (COSTAR 3513, Corning Incorporated, Corning, New York, USA) using PDMS (prepared from SYLGARD^® 184 silicone elastomer kit, Dow Corning, Midland, Michigan, USA) to allow later removal after biofilm had formed. For each experiment two rows of four modified wells were produced. For each row, three wells were prepared with two electrodes of the same PEDOT composite. For the remaining well, two polyester (Rosinco AB, Filipstad, Sweden) films shaped, washed, rinsed and dried similarly to the conducting composites were employed as non-conductive references. Biofilm devices were stored overnight at 70 °C before use.

The treatment followed to obtain native collagen was described in a previous work [26 (link)]. Collagen scaffolds with 30 wt. % HMW or LMW chitosan (on collagen dry basis) were prepared by freeze-drying. Hence, chitosan was dissolved in 100 mL of 0.5 M acetic acid under continuous stirring. Then, 5 g of collagen and 20 wt. % glycerol (on collagen dry basis) were added and the blends were maintained under mechanical stirring for 3 h at 125 rpm. Finally, the blends were poured into each well of a 12 multiwell plate (Costar 3513, Corning Incorporated), and the plate was frozen for 24 h at −23 °C and then freeze-dried (Alpha 1-4 LDplus freeze-dryer, CHRIST) for 48 h. Finally, cylinder-shaped chitosan/collagen samples (2.26 cm diameter and 1 cm height) were removed from the wells. Scaffolds were neutralized by immersion into a sodium hydroxide 0.4 M NaOH solution for 15 min and subsequently rinsed in water. In that way, amino groups of chitosan were deprotonated, leading to the disappearance of ionic repulsions and favoring physical crosslinking with collagen.

Internalisation of nanodiscs and liposomes was quantified by fluorescence measurement using Rh-DHPE-labelled lipid nanodiscs. HeLa cells were plated at a density of approximately 20 × 10⁵ cells per mL with 1 mL of culture medium in 12-well plates (Costar 3513, Corning). A 10 mM stock solution of lipid nanodiscs and liposomes with 1% Rh-DHPE was prepared and diluted with PBS to the desired concentration. The final concentration of lipid (DPPC) was set to 1.56 μmol L⁻¹ in the culture medium with HeLa cells. PBS was used as a negative control. After the exposure of HeLa cells to lipid nanodiscs or liposomes for 1 hour at 37 °C in 5% CO₂ atmosphere, the cells were washed 3 times with 1 mL of PBS to remove excess nanodiscs or liposomes. To obtain the suspension of cells, the adhered HeLa cells were trypsinized. Collected cells were centrifuged at 1500 rpm for 3 minutes and washed twice with PBS at 37 °C. Finally, the cells were resuspended in 1 mL PBS and the number of cells was counted using a Neubauer-improved cell counting chamber. The fluorescence intensity of cellular suspension was measured using a JASCO FP-8300 fluorescence spectrometer (Tokyo, Japan). Emission intensity at 585 nm was recorded with the excitation at 560 nm. Excitation and emission band-passes were set to 10 nm. The fluorescence intensity was normalised against 10⁴ cells.