Biguanide- and Oligo(Ethylene Glycol)-Functionalized Poly(3,4-Ethylenedioxythiophene): Electroactive, Antimicrobial, and Antifouling Surface Coatings

The challenge of infectious diseases remains a critical concern to the global public health. Recently, it is common to encounter touch-screen electronic devices everywhere to access services. The surface of such devices may easily get contaminated by an infected person, which leads to transmission of infectious diseases between individuals. Moreover, the challenge is complicated by surgical infections from implantable biomedical devices. Such problems can be minimized by the use of long-term active antimicrobial surface coatings. We present herein the preparation of novel electroactive antimicrobial surface coatings through the covalent attachment of the biguanide moiety onto 3,4-ethylenedioxythiophene (EDOT). The biguanide-functionalized EDOT (EDOT-BG) was thus electropolymerized on different substrates to give the corresponding poly(EDOT-BG) polymer. The poly(EDOT-BG) polymer showed an excellent bactericidal efficiency (∼92% bacterial death) and excellent biocompatibility with mammalian cells. Furthermore, the antimicrobial EDOT-BG was electro-copolymerized with antifouling tetra ethylene glycol functionalized-EDOT (EDOT-EG4) to give a multifunctional poly(EDOT-EG4-co-EDOT-BG) copolymer. The poly(EDOT-EG4-co-EDOT-BG) copolymer showed excellent resistance to protein adsorption and mammalian/bacterial cell binding without losing its bactericidal efficiency. These novel materials can be applied to domestic and bioelectronic devices to minimize infectious diseases.


Microorganisms and Growth Conditions
The antibacterial and antifouling study was performed using gram-negative Escherichia coli (E. coli). The bacteria were grown at 37 °C for 16 h at 180 rpm in nutrient broth (LB broth) (Difco, Detroit, MI) prepared in DI water. A 100 μL culture was taken from this and then added to 5 mL of fresh nutrient broth which was grown for 6 h at 37 °C and 180 rpm. From this culture, 1 mL culture was centrifuged at 10,000 rpm for 5 min. The obtained pellet was washed twice with PBS to remove excess medium and resuspended the pellet in PBS. 50 µL of 1 x 106 CFU/mL of bacterial concentration was used for further experiments using colony counting OD method.

Evaluation of Bactericidal Efficiency
To evaluate antibacterial activity, the bacterial culture was incubated with the samples on orbital shaker at 80 rpm /2 hours at 37oC. After the incubation, the samples were washed with PBS and antibacterial activities for all the samples were analyzed by recording the OD (600nm), plating method and Live/Dead Staining Assay.

Plating and OD (Optical Density) method
The microbial suspension treated with the samples was collected, and diluted in PBS, spread onto nutrient agar (LB Agar), and incubated at 37 °C for 18 h. 200 µL of suspension collected from the surface was used to record OD at 600nm for each sample. Bactericidal activities of the samples were determined by comparing colony counts (qualitatively) and OD between different samples.

Live/Dead Staining Assay
A live/dead staining assay was performed to evaluate the killing efficiency of the surfaces. The surfaces were incubated with E. coli bacteria (1 × 106 cells/mL in phosphate buffered saline (PBS), pH 7.4) for 2 hours at 37 °C, and then the surface was washed with sterile PBS. The surfaces were stained with a Live and dead bacterial assay kit (Thermofisher Scientific, Cat. L7007, a green-fluorescent nucleic acid stain which generally labels all bacteria and red-fluorescent nucleic acid stain which penetrates only bacteria with damaged membranes) for 15 min in the dark. After gently rinsing with sterile water and drying in air, the surface-attached bacteria were examined using a fluorescence microscope (IX71, Olympus, Japan) with a 40X objective lens. The 3 representative images were chosen for each surface. Three replicates were performed and the relative number of live (green) vs dead (red or yellow) bacteria were counted and the results are presented in mean ± SD.

MTT assay
In addition to live/dead cell viability assay studies, the cytotoxicity of biguanide containing PEDOTs (poly(EDOT-co-EDOT-BG) and poly(EDOT-BG)) were evaluated using the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Similar to live/dead cell assay method, HEK-293T cell lines were used for MTT assay studies. Bare ITO-glass and PEDOT were used as references. The cells were grown for 24 hours in DMEM culture media and the cytotoxicity study was evaluated according to the following procedure.
A 5 mg/mL MTT reagent was prepared and stored at 4 °C in dark until use. The stock solution of MTT was diluted 10-fold in the culture medium. To evaluate the in vitro cytotoxicity, the cell lines were seeded at a density of 1 × 104 cells/mL on 1 x 1 cm samples (bare ITO-glass, PEDOT, poly(EDOTco-EDOT-BG), and poly(EDOT-BG)) placed in 12-well cell culture plates and incubated for 24 hours. After incubation, the culture media was aspirated and cells were washed twice using pre-warmed PBS buffer. Next, 150 µL MTT reagent diluted in culture medium was added on top of the surfaces to be tested and incubated at 37 °C for 4 hours in a dark. MTT reagent was then removed from the culture medium followed by addition of 200 µL of DMSO to dissolve the purple crystals, and the plates were kept on shaking using a rotary shaker at 100 rpm for 15 min. For the reading, 200 µL solution was taken from each well and transferred to 96-well plates. Finally, the absorbances were measured at 570 nm. The DMEM medium was used as a blank. Cell viability was calculated using equation 1 and the % cell viability results are presented below: (1) Figure S2. MTT viability assay graph showing viability (%) of HEK-293T cells cultured on different substrates for 24 hours.

Antifouling property studies
A 50 µL of E. coli at a concentration of 10 6 cells/mL was pipetted onto each substrate. The sample was incubated at room temperature for 2 hours. Later, the sample was washed with PBS and stained with live and dead bacterial staining kit. The number of cells was determined with a (IX71, Olympus, Japan) mounted on Olympus IX81 fluorescent microscopy (Olympus, Japan) with 10X and 40x objective lens. Three separate samples were analyzed for each substrate.