Conjugation of Polysulfobetaine via Poly(pyrogallol) Coatings for Improving the Antifouling Efficacy of Biomaterials.

Antifouling treatment is critical to certain biomedical devices for their functions and patients' life. Facial, versatile, and universal coating methods to conjugate antifouling materials on a wide variety of biomaterials are beneficial for the fabrication of low-fouling biomedical devices. We developed a simple one-step coating method for surface conjugation of zwitterionic poly(sulfobetaine) via deposition of self-polymerized pyrogallol (PG). Poly(pyrogallol) could deposit copolymers of sulfobetaine methacrylate and aminoethyl methacrylate (pSBAE) on various biomaterials. pSBAE coatings inhibited as high as 99.8% of the adhesion of L929 cells and reduced protein adsorption significantly. The resistance against L929 cell adhesion was increased with increasing coating time and was positively correlated with the surface hydrophilicity and film thickness. Such a coating was robust to resist harsh sterilization conditions and stable for long-term storage in phosphate-buffered saline. We expect that the simple low-fouling pSBAE coating is applicable to the manufacture of medical devices.

Introduction

Surface deposition of antifouling polymers has been an effective strategy to reduce potential medical complications caused by undesirable biointerfacial interactions between medical devices and biological environment containing proteins and cells.[1] Antifouling polymers are often highly hydrophilic polymers, such as polysaccharides, poly(ethylene glycol) (PEG), and poly(zwitterions), that adsorb a great amount of water to form a barrier to reduce nonspecific interactions between biomaterials and biological systems.[2,3] The coating methods for surface conjugation of antifouling polymers are generally categorized into “graft-to” and “graft-from”.[4] “Graft-to” refers to physical or chemical immobilization of presynthesized polymers,[5−7] while “graft-from” refers to in situ polymerization of hydrophilic monomers from surface-bound initiators.[8−11] Some surface coating strategies, such as layer-by-layer (LbL) deposition,[12] chemical vapor deposition (CVD) of functional films,[13] and photoinitiated immobilization,[14] could be applied to a wide range of substrates, but possess certain disadvantages. LbL deposition is usually tedious, time-consuming, and could not survive harsh sterilization conditions. CVD requires expensive equipment. A facial, versatile, and universal method for depositing antifouling polymers to a wide variety of substrates is beneficial to the advancement of biomedical engineering.

Simple spontaneous “mussel-inspired” polydopamine (PDA) has been developed as a powerful surface coating technology.[15−18] PDA coatings possess strong and universal interfacial adhesiveness to substrates and chemical reactivity toward nucleophiles, such as amines and thiols.[19] We previously utilized the PDA’s unique characteristic to conjugate poly(ethylene imine)-g-PEG to several biomaterials for reducing nonspecific cell adhesion and protein adsorption.[20] Another spontaneous coating technology based on prebiotic chemistry inspired aminomalononitrile (AMN) polymerization has been developed in recent years.[21,22] Similar to the PDA coating, the AMN coating has been shown as a facile and versatile platform for immobilization of antifouling polymers.[23,24]

Many plant-derived compounds containing catecholic or galloyl moieties are also able to form strongly adherent coatings onto various substrates.[25,26] One advantage of polyphenolic compounds is their much lower costs in comparison to dopamine and AMN. Catecholic or galloyl groups are prone to oxidation into quinones or other complexes at mildly alkaline conditions in the presence of dissolved oxygen.[25] Oxidized polyphenols can oligomerize into higher-molecular-weight molecules[27] with lower solubility and inherent affinity toward various surfaces, ultimately leading to surface deposition.[28] Zwitterionic polymers tethered with catecholic or galloyl groups could be grafted onto various materials to provide a low-fouling property.[5,29] However, synthesis of catecholic or galloyl zwitterionic polymers is a tedious and complex task. Similar to the PDA reactivity toward amino groups,[28] polyphenolic coating could also be used for one-step deposition of antifouling materials.

The specific aim of this study is to develop one-step low-fouling coatings via the co-deposition of polysulfobetaine and pyrogallol. The mechanism of pyrogallol oligomerization postulated that galloyl molecules are oxidized and form dimers via oxidative intermediates of galloquinones. The galloyl dimers then undergo inter-gallol cross-linking to generate purpurogallin with a carbonyl group in a five-membered ring structure.[30] Molecules containing amines could be conjugated to purpurogallin via a ring-opening reaction with the carbonyl group. Therefore, galloyl molecules could be used as a mediator to conjugate amino molecules onto substrates. We synthesized copolymers of sulfobetaine methacrylate (SBMA) and 2-aminoethyl methacrylate (AEMA) and then deposited the copolymers on several types of materials via dip coating. AEMA provides amino groups for conjugation with galloyl molecules. The antifouling performance of the coatings was evaluated based on their resistance to cell adhesion and protein adsorption. The correlation between the antifouling efficacy and the surface properties of the coatings was investigated. The stability of the coatings was also studied.

Results and Discussion

Synthesis and Characterization of Poly(sulfobetaine methacrylate-co-2-aminoethyl methacrylate) (pSBAE)

A series of copolymers of SBMA and AEMA were synthesized with different monomer concentrations. The molecular weights of the copolymers, determined using GPC (Figure S1 in the Supporting Information), are listed in Table S2. The copolymers synthesized from 5 to 50 mol % AEMA possessed Mn = 104 610 with PD 10.7 and Mn = 31 535 with PD 1.64. The results showed that Mn of the copolymers was reduced with increasing AEMA. The positively charged AEMA might hinder polymerization due to electrostatic repulsion. We also noted decreasing PD of the copolymers with the addition of AEMA. Only pSBAE1 (AEMA 10%), pSBAE2 (AEMA 20%), and pSBAE5 (AEMA 50%) were used in the surface conjugation for antifouling experiments due to their low PD.

The compositions of pSBAE1, pSBAE2, and pSBAE5 were determined from the 1H NMR spectra (Figure S2 in the Supporting Information). The peak at 3.75 ppm arises from two protons adjacent to the quaternary amine of SBMA, whereas the peak at 4.2 ppm is ascribed to two protons adjacent to the oxygen atom of AEMA.[31,32] The ratios of the integral areas of the two peaks were used as the molar ratios of SBMA to AEMA in the copolymers. The molar ratios of SBMA to AEMA in pSBAE1, pSBAE2, and pSBAE5 were calculated as 10.37, 4.97, and 2.27, respectively. The AEMA molar percentages are thus estimated as 9.6, 20.1, and 44.1%, respectively, close to the AEMA compositions in the feed (Table S1 in the Supporting Information).

Deposition of PG/pSBAE Coatings

Polydimethylsiloxane (PDMS) was incubated in PG solution (8 mg/mL) for 24 h. Brown stains appeared on the PG-treated PDMS (Figure B). The samples further turned dark brown after incubation in silver nitrate solution (Figure C), indicating the reduction of silver ions on the PG coatings.[28] This phenomenon demonstrates successful PG deposition on PDMS. No obvious brown stain was found on the PDMS treated with PG/pSBAE1 (8/40 mg/mL) (Figure D), but the sample turned dark brown after immersion in silver nitrate solution (Figure E). The result indicates that the PG/pSBAE1 substrate was still able to reduce silver ions.

Figure 1

PDMS samples were treated with PG (8 mg/mL) or PG/pSBAE1 (8/40 mg/mL) for 24 h. (Top) Photos of PDMS samples: (A) pristine PDMS, (B) PG-treated PDMS, (C) PG-treated PDMS after immersion in 0.1 M silver nitrate, (D) PG/pSBAE1 PDMS, and (E) PG/pSBAE1 PDMS after immersion in 0.1 M silver nitrate. (Bottom) ATR-FTIR spectra of pristine, PG-treated, and PG/pSBAE1-treated PDMS.

The deposition of PG/pSBAE1 on PDMS was further demonstrated by ATR-FTIR (Figure , bottom). In comparison to pristine PDMS, the PG-deposited surface revealed an adsorption peak at 1644 cm–1, attributed to the C=C stretching vibration of PG’s aromatic ring, and a peak at 3424 cm–1, attributed to the O–H stretching vibration of PG. The PG deposition with pSBAE1 generated peaks at 1725, 1410, and 1122 cm–1, ascribed to the vibrations of C=O, S=O, and C–N stretching vibrations, respectively. The FTIR spectra demonstrate the deposition of pSBAE1 on PDMS together with PG.

Resistance of PG/pSBAE Coatings to L929 Cell Adhesion

The adhesion of L929 cells was first evaluated on a series of PG/pSBAE-coated tissue cell polystyrene (TCPS) (Figure A). The deposition of PG/pSB (without AEMA) decreased cell adhesion to 1.43 × 104 cells/cm2 from 2.98 × 104 cells/cm2 on TCPS. Incorporation of pSBAE further decreased cell attachment to much lower levels. The adhesion of L929 cells was almost inhibited on the TCPS coated with pSBAE1 (<0.13%), while the reduction in cell adhesion on the samples coated with pSBAE2 or pSBAE5 was approximately 85 or 75%, respectively, in comparison to pristine TCPS.

Figure 2

(A) Adhesion of L929 cells on TCPS coated with a series of PG/pSBAE with different AEMA molar ratios. The concentration of PG and the copolymers were 8 and 40 mg/mL, respectively. (B) Adhesion of L929 cells on TCPS coated with a series of PG/pSBAE1 with different pSBAE1/PG weight ratios. The concentration of PG was fixed at 8 mg/mL. L929 cells were seeded at a density of 2 × 104 cells/cm2 and cultured at 37 °C for 24 h. All values represent mean ± standard deviation; n = 4; * represents p < 0.001 vs TCPS and PG.

The PG/pSB-modified surface possessed partial resistance to L929 cells, suggesting that the modified surface might contain pSB even though pSB does not contain primary amines. We guess that pSB might be trapped in PG deposition. Nevertheless, the AEMA moiety is important for conjugation of pSBAE to enhance cell resistance. pSBAE1 possessed the best cell resistance among the three copolymers. The result suggests that a high AE ratio is unfavorable to the cell resistance, probably owing to the increase in positive charges of the copolymers. Since the pSBAE1 coating possesses the best cell-repellant ability among the three copolymers, pSBAE1 was used in the subsequent experiments.

We next evaluated the dependence of the weight ratios of pSBAE1/PG from 0.5 to 8 on the capability of anticell adhesion. When the pSBAE1 content was lower than or equal to the PG content (0.5 and 1), the cell attachment was reduced to ∼10% (Figure B). When the amount of pSBAE1 was 2-fold of the PG content, the cell attachment was reduced to only 1.5% of that on TCPS. When the content of pSBAE1 was increased to 5 times of PG, the cell adhesion was almost completely inhibited (<0.12%). It seems that a weight ratio of 5 is adequate to inhibit L929 cell adhesion, so the ratio was used in the subsequent experiments.

Poly(pyrogallol) possesses high affinity toward a wide variety of substrates such as metals, ceramics, and polymers,[25,28] so PG/pSBAE1 should be able to adhere to various materials. Several commonly used materials, such as PDMS, silicon (Si), polyethyleneterephthalate (PET), polystyrene (PS), glass, polyurethane (PU), and poly(vinylidene fluoride) (PVDF), were investigated in this study for the feasibility of the low-fouling coatings. Most of the substrates modified with PG/pSBAE1 possessed static water contact angles from 30 to 60° (Figure A). The reduction in the water contact angles in comparison to the counterpart the pristine materials indicates that all of the materials become more hydrophilic after PG/pSBAE1 coatings. The L929 cell adhesion was almost inhibited on all of the modified substrates (<100 cells/cm2 except Si and PU <300 cells/cm2, Figure B). The results demonstrate the applicability of PG/pSBAE1 coatings to several types of substrates.

Figure 3

PG/pSBAE1 (8/40 mg/mL) was coated on several substrates, including PDMS, silicon, PET, PS, glass, PU, and PVDF, for 24 h. (A) Static water contact angle measurement. At least 10 spots of each sample were measured. (B) L929 cells were seeded at a density of 2 × 104 cells/cm2 on each modified sample (n = 4) and cultured at 37 °C for 24 h. Value = mean ± standard deviation; * represents p < 0.001 between the pristine and modified substrates.

Dependence of Resistance to L929 Cell Adhesion on PG/pSBAE1 Coating Time

The correlation between the surface properties and the cell resistance efficacy of PG/pSBAE1 coatings was next evaluated. A series of PG/pSBAE1 coatings were fabricated on PDMS by varying the deposition time from 0.5 to 24 h. The deposition of PG/pSBAE1 was characterized using XPS (Table ). We expect that the deposition of pSBAE1 results in the appearance of nitrogen and sulfur atoms since sulfur and nitrogen atoms only exist in pSBAE1. Indeed, after half an hour coating, the N and S peaks appeared in the XPS spectra and the intensities of the two atoms increased from 0.5 to 3 h. The sulfur and nitrogen contents were not changed significantly after 3 h coating. The Si content in XPS was also decreased with PG/pSBAE1 deposition. However, Si still appeared in the XPS spectrum after 24 h coating.

Table 1

Surface Atomic Percentages of PG/pSBAE1 (8/40 mg/mL) Coated PDMS at Different Coating Time Intervals (0, 0.5, 1, 3, 5, 8, 12, 24 h)

	coating time (h)
elemental composition (atomic %)	PDMS	0.5	1	3	5	8	12	24
O 1s	27.23	27.05	25.64	27.99	26.45	26.26	25.71	26.47
N 1s	0	2.49	3.03	4.00	3.21	3.44	3.30	3.42
C 1s	48.69	54.37	56.94	54.44	56.73	57.00	58.25	57.88
S 2s	0	1.49	2.11	2.46	2.62	2.78	2.83	3.44
Si 2p	24.08	14.39	11.55	8.61	11.00	10.42	9.79	8.78

The morphology and surface roughness changes of PG/pSBAE1 coatings were determined using SEM and AFM. The SEM images show that PG seems to form tiny particles and aggregates on PDMS, and the aggregates were increased with increasing coating time intervals (Figure B). The deposition of PG/pSBAE1 resulted in aggregates and islets from 0.5 to 3 h, reflected by the increasing surface roughness (root mean square) from 1.4 to 87 nm (Figure S3). After 5 h of PG/pSBAE1 coating, the surface became smoother with decreased roughness (0.42 ± 0.03 nm), suggesting that PG/pSBAE1 coating gradually covered the whole substrates. The surface roughness reached a minimum (0.17 ± 0.01 nm) after 12 h coating but increased to 6.73 ± 0.1 nm after 24 h coating.

Figure 4

SEM images of (A) pristine PDMS and (B) PG (8 mg/mL)- and PG/pSBAE1 (8/40 mg/mL)-coated PDMS with different coating time intervals (0.5, 1, 3, 5, 8, 12, and 24 h).

The development of PG/pSBAE1 coatings was evaluated via the changes in surface wettability and film thickness. The water contact angles decreased from 93.8° at 0.5 h to the lowest ∼30° at 12 h and remained at a similar level at 24 h (Figure A). It should be noted that film thickness was determined on silicon, not on PDMS. The PG/pSBAE1 films grew roughly linearly from 12.8 nm at 0.5 h to 89.2 nm at 24 h (Figure A). The previous XPS data on the PDMS samples show that Si still appeared on the surface even after 24 h coating. However, the film thickness on silicon revealed that the film was too thick to find the underlying substrate. The possible reasons might be (i) the difference in the thickness of PG/pSBAE1 deposition on silicon and PDMS, (ii) uneven coatings on PDMS, or (iii) the migration of small molecules of monomers and oligomers from PDMS to the coatings. We feel that the possibility of the last one is high because it is one plausible mechanism for the hydrophobic recovery of PDMS after hydrophilic treatment.[33]

Figure 5

PG/pSBAE1 (8/40 mg/mL)-coated PDMS, except film thickness (silicone), with different coating time intervals. (A) Film thickness on silicon and water contact angles on PDMS at different coating times. At least 10 spots were measured for each sample. (B) L929 cells were seeded at 2 × 104 cells/cm2 on each sample (n = 4) and cultured at 37 °C for 24 h. (C) Correlation between cosine of water contact angle and cell attachment. (D) Correlation between film thickness and cell attachment. (E) Adsorption of fibrinogen from 0.1 mg/mL in PBS onto PDMS that was deposited with PG/pSBAE1 at different coating times (n = 4); “a.u.” represents arbitrary unit; value = mean ± standard deviation.

The adhesion of L929 cells was evaluated on the PG/pSBAE PDMS. The cell attachment was decreased with increasing coating times (Figure B). In comparison to the cell adhesion to PDMS, the number of the attached cells was decreased by 40% on the 1 h sample, while 70% cell adhesion was repelled on the 3 h sample. After 8 h coating, the cell attachment was reduced to merely 10%. Cell adhesion was reduced to ∼0.66% after 12 h coating. There is a slight increase in cell adhesion after 24 h coating.

The cell attachment was then correlated to the surface properties. First, cell attachment was correlated with water contact angle of the substrates (Figure C). The water contact angles were expressed by their cosine values. A cosine value close to 1 means perfect wettability, while a negative value represents a hydrophobic surface. It is apparent that the inhibitory efficacy for cell attachment was positively correlated to the wettability of the PG/pSBAE1 coatings. When the cosine value approached 0.9, the cell attachment was almost completely inhibited. The cell resistance of PG/pSBAE1 coatings was positively correlated to the film thickness (Figure D).

Fibrinogen Adsorption on PG/pSBAE1

Protein adsorption is another important aspect of biofouling.[34,35] In these studies, fibrinogen, the major plasma protein in mediating platelet adhesion and activation,[12,36] was used for the investigation of protein resistance of the PG/pSBAE1-modified PDMS. Fibrinogen adsorption was gradually decreased with increasing coating times (Figure E). The optimal condition for reducing fibrinogen adsorption was 24 h coating, at which 93% fibrinogen adsorption was inhibited.

Stability of the PG/pSBAE1 Coating

Stability of antifouling coatings is an important aspect for the clinical application of biomedical devices. In this study, the stability of the PG/pSBAE1 coating was evaluated according to its efficacy in cell resistance after long-term incubation in phosphate-buffered saline (PBS) or autoclave sterilization. The PG/pSBAE1 coating on PDMS retained its cell-repellent ability after 14 days of incubation in PBS (Figure A). Even after 21 days, the surface retained the low cell attachment. The PG/pSBAE1 substrate that was autoclaved still retained the very low cell attachment (Figure B). We did not find any significant change in water contact angles on the PG/pSBAE1 coatings after long-term incubation or autoclaving, suggesting the preservation of the PG/pSBAE1 coating on PDMS. Although the stability of PG/pSBAE1 coatings on other types of substrates remains to be investigated further, the data show the potential applications of our conjugation method on biomedical devices.

Figure 6

Stability of anticell adhesion efficacy of the PG/pSBAE1 coating on PDMS. L929 cells were seeded at 2 × 104 cells/cm2 on each sample (n = 4) and cultured at 37 °C for 24 h. (A) Samples were incubated in PBS for a prolonged period. (B) Samples were autoclaved. Value = mean ± standard deviation, n = 4.

The coating method based on pyrogallol chemistry has several advantages. First, the fabrication process is easy and simple, even for a person without practical experience in a chemical laboratory. Second, polyphenols, e.g., pyrogallol, are much cheaper than dopamine and AMN, both performing similar deposition. Third, PG coatings on PDMS are very stable for long-term storage and resist stringent sterilization processes such as autoclaving. Such a property is rarely demonstrated in other antifouling treatment. Finally, PG/pSBAE coating looks transparent. Such a property is beneficial to the applications, such as sensing and intraocular lens, which need high light transmittance. Therefore, we believe that the coating method has a high potential for broad application to biomedical devices.

Conclusions

This work developed a simple one-step organic coating for surface conjugation of antifouling polysulfobetaine on various substrates. Poly(pyrogallol) anchors pSBAE to substrate via the AE moieties. The results proved that the PG/pSBAE coatings resist the adhesion of L929 cells and reduce protein adsorption greatly. The antifouling efficacy of the PG/pSBAE coatings was positively correlated with surface wettability and film thickness. The PG/pSBAE coating on PDMS could resist harsh sterilization environment and long-term storage. We expect that the PG-based coating is suitable for medical devices that require a low-fouling surface.

Materials and Methods

Materials

Chemicals

Sulfobetaine methacrylate (SBMA, cat#537284) was purchased from Taiwan Hopax Chems (Kaohsiung, Taiwan). 2-Aminoethyl methacrylate hydrochloride (AEMA, cat#516155), azobisisobutyronitrile (AIBN), dimethyl sulfoxide (DMSO), pyrogallol (PG, cat#P0381), and trypan blue were bought from Sigma-Aldrich. Gentamycin, 10× trypsin–EDTA, Fungizone, and minimum essential medium alpha medium (α-MEM) were purchased from GIBCO. Fetal bovine serum (FBS) was purchased from JRH Biosciences (Australia). The cell culture medium was composed of α-MEM supplemented with 10% FBS, 10 mL of fungizone, 5 mL of gentamycin, and 0.4 mL of 2-mercaptoethanol in 1 L. AlexaFluor 488-labeled fibrinogen (AF-Fib) was purchased from Thermo Fisher.

Substrates

Tissue cell polystyrene (TCPS) was obtained from Nunc. Polydimethylsiloxane (PDMS; Sylgard 184) was obtained from Dow Corning. Polyethyleneterephthalate (PET) and polystyrene (PS) were received from Nihon Shiyaku Industries Ltd. (Japan). Silicon wafer was obtained from Yia Chuan Company (Taoyuan, Taiwan). Polyurethane (PU) and poly(vinylidene fluoride) (PVDF) substrates were fabricated using solution casting, as described in the Supporting Information.

Synthesis and Characterization of Poly(SBMA-co-AEMA) (pSBAE)

A series of copolymers of SBMA and AEMA were synthesized by free-radical polymerization (Scheme ). The amount of SBMA was fixed at 0.8496 g (3 mmol) with the addition of AEMA at a molar ratio of 10, 20, and 50% of SBMA, and the as-formed copolymers were referred to as pSBAE1, pSBAE2, and pSBAE5, respectively, as listed in Table S1 (Supporting Information). SBMA and AEMA were dissolved in deionized water to the desired concentrations and then mixed with 0.005 g (0.03 mmol) AIBN in DMSO, followed by nitrogen purge. Polymerization was initiated by elevated temperature (70 °C). After 20 h reaction, the products were dialyzed against deionized water for removal of unreacted monomers or oligomers and then freeze-dried. The compositions of the synthesized copolymers were verified by 1H nuclear magnetic resonance (Bruker AVIII HD 400 NMR, Germany). Molecular weights of the copolymers were determined by gel permeation chromatography (Jasco, UV-2075, Japan).

Scheme 1

Polymerization of the Copolymer of SBMA and AEMA

Surface Deposition of PG/pSBAE

PG in phosphate-buffered saline (PBS, pH 7.4) was mixed with the same volume of pSBAE in PBS to the desired concentrations. The mixtures were added onto substrates and then incubated at 45 °C with constant agitation for a period of time. The substrates were rinsed with deionized water and then air-dried.

Characterization of PG/pSBAE Coatings

FTIR spectra of the pSBAE coatings were collected using a Fourier transform infrared spectrometer (Spectrum 100, Perkin Elmer) over 16 scans in the region of 1000–4000 cm–1 using attenuated total reflectance mode. The XPS analysis was performed using an AXIS Ultra DLD spectrometer with a monochromated Al Kα source at a power of 45 W (15 kV × 3 mA), a hemispherical analyzer operating in the fixed-analyzer transmission mode, and the standard aperture (1 mm × 0.5 mm slot). The total pressure in the main vacuum chamber during analysis was reduced to 10–8 mbar. Each specimen was analyzed at a takeoff angle of 72.5° as measured from the horizontal surface. Thus, the analytical depth value of XPS ranges between 5 and 10 nm from the top surface. An elliptical area with approximate dimensions of 0.3 mm × 0.7 mm was analyzed on each sample.

The surface morphology of the coatings on silicon was analyzed using a field emission scanning electron microscope (FESEM, NovaTM NanoSEM 230, Scanservice). The surface topography and roughness of the coatings were measured using an atomic force microscope (NanoScope IIIa, Digital Instruments). The root-mean-square roughness values were calculated from 500 nm × 500 nm images, n = 3.

Surface wettability was evaluated via static contact angle measurement of deionized water (5 μL) using a contact angle system (FTA125, First Ten Angstroms) at room temperature. At least 10 spots were measured for each sample. The thickness of coatings was determined on silicon based on spectral reflectance (Model F20, Filmetrics). At least 10 points were measured for each sample.

Evaluation of Fibrinogen Adsorption on PG/pSBAE-Coated PDMS

Fibrinogen adsorption to PG/pSBAE-coated PDMS was evaluated using total internal-reflection fluorescence.[37] Briefly, AF-Fib in PBS (0.1 mg/mL) was incubated on the modified PDMS samples (1 mL/sample) at room temperature for 30 min, followed by rinsing with PBS to remove loosely bound AF-Fib. Surface fluorescence was visualized and recorded using a fluorescence microscope (Leica DM6000B Upright/OLYMPUS IX71 Inverted Microscope System). For each sample, 10 images were randomly captured from the samples and the fluorescence intensities were analyzed using NIH ImageJ software. The fluorescence intensities on the modified PDMS were in comparison to that on the pristine PDMS.

Cell Adhesion to the PG/pSBAE Modified Surfaces

TCPS deposited with PG/pSBAE was sterilized by exposure of UV for 2 h, followed by immersion in 70% ethanol for 20 min. L929 cells were seeded at a density of 2 × 104 cells/cm2 on each sample and maintained in a 37 °C incubator supplied with 5% CO2 for 24 h. Cell numbers were quantified by counting attached cells from phase microscopy images after gentle washing with PBS twice except silicon due to opacity. Cell numbers on silicon were counted using a fluorescence microscope after cell nuclei were stained with 4′,6-diamidino-2-phenylindole. Five images were randomly taken from each sample (four samples per substrate).

Stability Test

The stability of the pSBAE/PG coatings on PDMS was evaluated according to the changes in cell resistance of the substrates after (i) immersion in PBS at room temperature with continuous shaking at 50 rpm for several days or (ii) autoclaving for 1 h (122 °C, 1.2 kg/cm2).

Statistical Analysis

The data were reported as mean ± standard deviation (SD). The statistical analyses between different groups were determined using Student’s t test. Probabilities of p ≤ 0.05 were considered a significant difference. All statistical analyses were performed using GraphPad Instat 3.0 program (GraphPad Software, La Jolla, CA).