Integrated zwitterionic conjugated poly(carboxybetaine thiophene) as a new biomaterial platform

An integrated zwitterionic conjugated polymer-based biomaterial platform was designed and studied to address some of the key challenges of conjugated polymers in biomedical applications.


S4
After the removal of methanol, the crude product was re-dissolved in diethyl ether, washed with DI water and dried with anhydrous magnesium sulfate. Pure product was obtained after filtration and evaporation of solvent. Structure was confirmed with 1 H NMR and the data was in agreement with the previous report.

Synthesis of homo-polymers and copolymers:
Homo-polymer P1. 6.11 g (37.7 mmole) of anhydrous FeCl 3 was suspended in 60 mL of anhydrous chloroform under a positive nitrogen flow. The mixture was cooled in an ice-bath (0 o C) and kept agitated for 30 minutes. 2.0 g (9.42 mmol) of compound 2 dissolved in 30 mL of dry chloroform was slowly added into the mixture during a period of one hour. Then the reaction was stirred for 24 hours at room temperature under nitrogen. After the reaction, the productwas washed with chloroform and dried with rotary evaporator. Then itwas redissolved in DI-water and purified through dialysis withcellulose dialysis membrane (1 k cut off). Water was changed daily for a week, and the solution was lyophilized to obtain P1 at 20 % yield. 1 H NMR (300 MHz, D 2 O) δ 6.6-7.6 (m, thiophene ring proton, 1H), 3.0-4.3 (m, thiophene ring -CH 2 -and -NH-

Hydrogel preparation
Both PCBTh-co-ThMAA and PCBTh-co-ThRGD hydrogels were prepared via similar thermo-initiated polymerizations as follows. 100 mg of copolymers was dissolved in 400 μL aqueous solution with 0.5 wt % of thermo-initiator (VA-044). Then the solution was transferred into a mold made of two quartz slides separated by a1 mm thick PTFE spacer and polymerized at 50 o C for overnight. The gel was equilibrated in DI water and water was changed daily for 7 days. The wet weight of the hydrogel samples was measured after the removal of excess water. PThAA hydrogel was prepared according to a reported method and used as a control in this study. [3]

Polymer film preparation
Polymer thin films were prepared with agraft-to method. Copolymer P8 (PCBTh-co-ThSH) with free thiol end group was prepared at the concentration of 10 mg/mL in a mixed solvent S9 of 90% DI-water and 10% methanol by volume. 400 µL of polymer solution was drop-casted on a gold-coated SPR chip. It was put in a petri-dish and left undisturbed until solvent evaporated at room temperature. Sample was washed with PBS and dried with filtered air before the SPR measurement.

Electrochemical study
The AC impedance spectrum was measured by a Solartron Model 1260 Impedance/Gainphase Analyzer with a Model 1287 potentiostat/galvanostat in the frequency range from 0.1 mHz to 100 kHz at low amplitude voltage (~10 mV) [22]. The hydrogel sample were cut into a disc with a diameter of 6.8 mm and put between to stainless steel electrodes. The ionic and electronic conductivities of hydrogels were calculated with a previously reported method. [4] The ionic resistance, Ri, can be determined from the relationship 1/R1 = 1/Ri +1/Re, where R1 is the high-frequency semi-circle resistance from impedance data and Re is the electrical resistance measured under small applied DC potentials (-30 mV -+30 mV) using the potentiostat. Cyclic voltammetry (CV) can provide potentiodynamic electrochemical measurements and stability measurement. [5] Fig.S9 shows CV curves and the impedance curve and of PCBTh-co-ThMAA hydrogel based electrodes using a two electrode system. Fig.S9A shows the rate-dependent CVs with the potential window of 0 to 1 V at scan rates of 5, 10, 20, 30 and 50 mV/s. CV were recorded in the potential range of 0-1 V using the potentiostat.The complex diagram shows a lineal behavior at low frequencies, which indicatesthatthe mass transport is the dominant mechanism. The capacitive response at medium frequencies denotes the current carries within the material.

Protein adsorption study
Protein adsorption study: A custom-built four-channel SPR sensor was used to measure protein adsorption on pCBTh-co-ThSHcoated surface. Firstly, PBS solution at 50 µL min -1 flow rate was used to obtain a baseline signal. 1 mg mL -1 of Fg solution and1 mg mL -1 of BSA were then injected into different channels for 10 minutes followed by a PBS wash to remove any loosely bound proteins. The amount of adsorbed proteins was calculated as the change in wavelength before and after protein injection.

7.Cell adhesion study
BAECs were chosen to study cell adhesion on hydrogel surfaces, following a similar procedure as in a previous work. [6] Hydrogel samples were equilibrated in DI-water and then transferred tosterilized PBS, exposed under UV for half an hour before the experiment.
BAECs were seeded on different hydrogelandcontrolsurfaces at a concentration of 10 5 cells mL -1 in DMEM containing 10% FBS and 1% penicillin-streptomycin, and kept in an incubator with 5% CO 2 at 37 o C for 24 hours. After the incubation,medium was removed from the wells and changed to the staining solution that prepared in sterilized PBS as follows.
Fluorescein diacetate was dissolved at a concentration of 10 mgmL -1 in acetone, then50 µL of the solution was diluted in 10 mL sterilized PBS and used for stainingthe cells. After incubated for 5 min with the staining solution, surface cell coverage and cell morphology was visualized and imaged with anOlympus IX70fluorescence microscope equipped with a FITC filter at ×10 magnification.

Water content measurement
The water content is a basic property of hydrogel materials for biomedical applications. The wet weight of the hydrogel sample was measured after the removal of excess water. Dry weight was recorded after the samples had been freeze-dried for 48 hours. The water contents of hydrogels (Table s1) are calculated by (Wet weight -Dry weight)/Wet weight x 100%.

Cytotoxicity study
The cytotoxicity of the zwitterionic polymer was studied with various concentrations of pCBTh. 100 μL of BAEC cells solution, at a concentration of 10 5 cellsmL -1 , were incubated in a 96 well plate for 24 hours with differentconcentrations (0.5, 5x10 -2 , 5x10 -3 , 5x10 -4 and5x10 -5 mgmL -1 ) of pCBTh. 6 replicates were used for each concentration. As a control, the same cells were also incubated at the same conditions without adding pCBTh. After 24 hours incubation, cells were stained with the same method as discussed in cell adhesion study.
Representative fluorescence imagesof surviving cells were taken for each condition (Figure S11 S10), with an Olympus IX70 fluorescence microscope equipped with a FITC filter at ×10 magnification. The number of cells was counted by three replicates andrelative viability was calculated and summarized in Figure S11. Figure S10. Representative fluorescence images of BAECs treated with a series of dilutions of pCBTh polymer A) 0.5 mgmL -1 , B) 5x10 -2 mgmL -1 , C) 5x10 -3 mgmL -1 , D) 5x10 -4 mgmL -1 , E) 5x10 -5 mgmL -1 and E) untreated cells, after 24 hours incubation in DMEM medium. Figure S11. Representative cytotoxicity assay of BAECs treated with a series of dilutions of pCBTh polymer in culture media, expressed as a percentage of control untreated cells S12

Optical properties study
The UV-vis absorption spectra of pCBTh were collected on a Hewlett Packard 8453 UVvisspectrophotometer. Samples were prepared in 20 mM PBS buffer solution at different pH, from pH 2 to pH 12. Fluorescence emission spectra were collected on a PerkinElmer LS 55 fluorescence spectrometer, excited at 411 nm. Figure S12. Normalized UV-vis spectra of pCBTh in 20 mM phosphate solution at different pH values.