A robust thin film surface with a selective antibacterial property

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Applications and Health

A robust thin film surface with a selective antibacterial property enabled via a cross-linked ionic polymer coating for infection-resistant medical applications Goro Choi, Gu Min Jeong, Myung Seok Oh, Munkyu Joo, Sung Gap Im, Ki Jun Jeong, and Eunjung Lee ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00241 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 2, 2018

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ACS Biomaterials Science & Engineering

A robust thin film surface with a selective antibacterial property enabled via a cross-linked ionic polymer coating for infection-resistant medical applications Goro Choi‡, Gu Min Jeong‡, Myung Seok Oh‡, Munkyu Joo, Sung Gap Im, Ki Jun Jeong*, Eunjung Lee* Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Daejeon 34141, Republic of Korea. KEYWORDS antibacterial coating, initiated Chemical Vapor Deposition (iCVD), crosslinking, ionic polymer, device-associated infections.

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ABSTRACT The fabrication of new antibacterial surfaces has become a primary strategy for preventing device-associated infections (DAIs). Although considerable progress has recently been made in reducing DAIs, current antibacterial coating methods are technically complex and do not allow selective bacterial killing. Here, we propose novel anti-infective surfaces made of a cross-linked ionic polymer film that achieve selective bacteria killing while simultaneously favoring the survival of mammalian cells. A one-step polymerization process known as initiated chemical vapor deposition (iCVD) was used to generate a cross-linked ionic polymer film from 4vinylbenzyl chloride (VBC) and 2-(dimethylamino) ethyl methacrylate (DMAEMA) monomers in the vapor phase. In particular, the deposition process produced a polymer network with quaternary ammonium crosslinking sites, which provided the surface with an ionic moiety with an excellent antibacterial contact-killing property. This method confers substrate compatibility, which enables various materials to be coated with ionic polymer films for use in medical implants. Moreover, the ionic polymer-deposited surfaces supported the healthy growth of mammalian cells while selectively inhibiting bacterial growth in co-culture models without any detectable cytotoxicity. Thus, the cross-linked ionic polymer-based antibacterial surface developed in this study can serve as an ideal platform for biomedical applications that require a highly sterile environment.


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INTRODUCTION Rapid advances in medical and healthcare systems have led to growing interest in antibacterial surface coatings because bacterial infections from biomedical devices remain an urgent issue. Device-associated infections (DAIs) have become a serious issue due to the increased risk of infectious diseases that require hospitalization.1-2 In the USA, 722,000 DAI cases resulted in approximately $11 billion of medical expenses, and DAIs were responsible for nearly 75,000 deaths in 2011.3 DAIs usually arise from the irreversible steps of bacterial adhesion, colonization, and biofilm formation on the surfaces of medical devices.4-5 Altering the surface properties of medical devices is a simple and direct approach to prevent DAIs, and extensive research efforts have focused on this objective.6-8 Several surface modification methods such as non-adhesive, antibiotic-releasing, silver-coating, or direct killing approaches have been developed and evaluated for their ability to prevent bacterial infections on implant surfaces. For example, polyethylene glycol (PEG)-coated surfaces exhibit bacteriarepellent properties.9 Based on the antibiotic-releasing method, several antibacterial agents such as chlorhexidine,10 gentamicin11 and silver nanoparticles12 have been incorporated into the surfaces of devices and released to target sites. Various cationic compounds containing quaternary ammonium compounds (QACs),13-17 chitosan derivatives,18 and antibacterial peptides (AMPs)19 are reportedly capable of killing bacteria through direct interactions with bacteria, allowing them to penetrate the bacterial cell membrane. However, these methods are characterized by inherent limitations of the solution-coating process, such as complicated, laborious procedures, a lack of controllability and leaching problems.20-21 Antibiotic release methods must overcome these limitations, such as the difficulty in ensuring controlled antibiotic


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release, and the initial burst and depletion of antibiotic agents. However, silver-based bactericidal surfaces have been shown to induce cytotoxicity side effects.13,22 In addition, silverbased bactericidal surfaces are only applicable to limited substrate materials with specific surface properties. Surface modifications of medical devices for clinical applications should selectively favor host tissues while preventing bacterial infections.14-15 The adhesive property of bacteria and host cells on some surfaces is an important issue that must be considered in designing antibacterial surfaces. According to the “race for the surface” concept,16 if tissue cell integration succeeds over bacterial adhesion in the competition to cover the implant surface, then bacterial adhesion and colonization can be effectively hindered. A previous experimental methodology proved this concept that mammalian cells can attach before the adhesion of bacterial adhesion in co-culture studies, and thus, mammalian cells can displace primary bacterial colonization.17-18 Otherwise, the bacterial adhesion and colonization markedly reduces the accessibility of mammalian cells to the surface, which leads to severe bacterial infection. In addition, the 6-h post-implantation period is known as the “decisive period” during which the long-term success of the implant is expected if host cell attachment supercedes bacterial adhesion.19,23 Therefore, antibacterial surfaces to selectively kill bacteria over mammalian cells have received growing interest, although modification methods are rarely reported. In this report, we developed a facile but robust surface modification method to synthesize a cross-linked ionic polymer film on biomedical substrates for infection-resistant and host cellcompatible surfaces. This innovative approach is performed using a solvent-free polymerization process that incorporates a chemical vapor deposition (iCVD) process, which does not generate any chemical contamination during deposition. This process also enables precise control over the


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surface chemical composition of the polymer. Moreover, due to the elaborate synthesis scheme, the ionic crosslinking reaction occurs simultaneously with the polymerization reaction in a onestep manner, resulting in a highly durable surface and minimizing substrate damage.24-25 A series of cross-linked ionic copolymers were synthesized from two monomers, vinyl benzyl chloride (VBC) and 2-(dimethylamino)ethyl methacrylate (DMAEMA), to yield a homogeneous copolymer film of p(VBC-co-DMAEMA) with ionic quaternary ammonium chloride (-NR3+Cl-) functionalities and excellent antibacterial properties. X-ray photoelectron spectroscopy (XPS) analysis and a bacterial killing assay revealed that the antibacterial efficiency increased with increasing amounts of quaternary ammonium groups in the copolymers. The ionic copolymer exhibited a total bacterial killing effect against both Corynebacterium glutamicum (C. glutamicum, a gram-positive bacterium) and Escherichia coli (E. coli, a gram-negative bacterium). Furthermore, various types of substrate materials such as polyethylene terephthalate (PET),26 polydimethylsiloxane (PDMS),27 and titanium28 successfully acquired antibacterial activities upon coating with the ionic copolymer. The selective antibacterial property of the modified surfaces was also investigated through the co-culture of bacteria and mammalian cells, demonstrating that the copolymer film-coated surface encouraged mammalian cell growth and function while retaining bacterial killing activity.

RESULTS AND DISCUSSION 1. Fabrication of cross-linked ionic copolymer films Antibacterial surfaces were generated by depositing a cross-linked ionic copolymer film onto the target substrate. A series of cross-linked p(VBC-co-DMAEMA) copolymers were synthesized via iCVD with two monomers, namely, 2-(dimethylamino)ethyl methacrylate


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(DMAEMA) and 4-vinylbenzylchloride (VBC) monomers.29 iCVD allows polymer thin films to form on various substrates in which a gas-phase initiator takes part in the radical polymerization of monomers. Because iCVD is a solventless process, surface damage imposed by the surface coating process can be minimized. Minimizing the surface damage makes this process suitable for surface modifications of temperature- and solvent-sensitive substrates, particularly for biomedical devices.30-31 This facile one-step method enables a two-in-one process during which crosslinking polymerization and thin film formation occur simultaneously on the substrate. A schematic illustration of the copolymer film deposition procedure using two monomers, VBC and DMAEMA, is shown in Figure 1a. The degree of crosslinking was modulated simply by varying the flow rate of VBC and DMAEMA, which resulted in the precise control of the surface content of quaternary ammonium moieties within the copolymer network. The feed ratios of VBC to DMAEMA were adjusted to 2:1 (V2D1), 1:1 (V1D1), 1:2 (V1D2), and 1:3 (V1D3) (Table S1 in Supporting Information (SI)). Peaks representing the C-Cl bond in VBC and the carbonyl (C=O) bond in DMAEMA were observed at 672 cm-1 and 1722 cm-1, respectively, in the FT-IR spectra (Figure 1b). The peak intensity of each functionality changed as the VBC/DMAEMA feeding ratio was altered. From pV2D1 to pV1D3, VBC content decreased while DMAEMA content increased, and correspondingly, the intensity of the C-Cl peak decreased while the intensity of the C=O peak increased. The vinyl peak (C=C) from the VBC monomers at 1630 cm-1 (stretching) and 908 cm-1 (bending) also disappeared after polymerization. During copolymer deposition process, the tertiary amines of DMAEMA were partially converted into quaternary ammonium compounds via a crosslinking reaction with the chloride moiety in VBC. A broad band at 3377cm−1 correspond to hydroxyl functionality which is resulting from the binding of water molecules on the quaternary ammonium groups in p(VBC-


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Figure 1. (a) Schematic of antibacterial surface modification via cross-linked ionic polymer coating and structure of p(VBC-co-DMAEMA) copolymer. (b) FT-IR spectra of a series of p(VBC-co-DMAEMA), pV2D1, pV1D1, pV1D2, pV1D3. (c) XPS survey scan spectra of pV1D3. High-resolution scan of N1s(right-top) and Cl2p(right-bottom). (d) XPS high-resolution scan of N 1s of pV2D1, pV1D1, pV1D2 and pV1D3. The blue and grey regions represent N+ peak and N peak, respectively.

co-DMAEMA) copolymers. Furthermore, a new peak binding energy of 402 eV attributed to the quaternization of the tertiary amine in DMAEMA was observed by XPS (Figure 1c and 1d).


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These results demonstrated that the two monomers were successfully copolymerized at various controlled compositions via iCVD. Because the quaternary ammonium moiety is a key component of the antibacterial property, the atomic composition of quaternary ammonium (N+) within the series of the synthesized copolymer films were investigated by XPS (Figure 2a). Due to the low stability of homopolymers, such as the high water solubility of pDMAEMA and the delamination of pVBC in aqueous solution, copolymer films were only utilized in this study for surface modifications. The fraction of DMAEMA increased from 14.73% for pV2D1 to 69.33% for pV1D3 with the input flow rate of DMAEMA monomers. With the increased proportion of DMAEMA from pV2D1 to pV1D3, the atomic composition of total N increased from 0.75% to 3.78% (Table S1). The fraction of quaternary ammonium (N+) in the copolymer film was maximized when the feed ratio of VBC to DMAEMA was 2:1.The calculated crosslinking degree of the copolymer increased to 45.75% for pV1D2 and then slightly decreased to 35.12% for pV1D3. The difference between the feed ratio and the resulting compositional ratio of monomers in the copolymers is due to the vapor pressure difference between the monomers. The vapor pressures of DMAEMA and VBC are ~600 mTorr at 25°C and ~100 mTorr at 25°C, respectively. To synthesize pV1D3, a relatively large amount of DMAEMA was introduced compared to the amount of VBC, which led to a reduction in the crosslinking degree by the early depletion of VBC, subsequently resulting in reduced content of quaternary ammonium in the pV1D3 copolymer. The change in the hydrophilicity of the copolymer films with various quaternary ammonium compositions was determined by measuring the zeta potential and the water contact angle (Figure 2b). The zeta potential analysis revealed that the positive surface charge of the


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copolymer films increased from 1.26 mV for pV2D1 to 22.05 mV for pV1D2. The surface charge was saturated with the maximum crosslinking degree, and the zeta potential of pV1D3 was 21.36 eV. The trend in the zeta potential paralleled that of the atomic ratio of quaternary ammonium (N+), indicating that the increase in the positive surface charge of the copolymer film was attributed to the enriched content of the quaternized amine moiety resulting from the crosslinking process between VBC and DMAEMA. As expected, the static water contact angle decreased with increasing content of DMAEMA from 73.5° for pV2D1 to 51.2° for pV1D3, demonstrating that the copolymer films exhibited more hydrophilic properties with increasing DMAEMA fractions (Figure S1 in Supporting Information). In addition, the copolymer film was fabricated homogeneously onto the target substrate without any phase segregation (Figure S2 in Supporting Information). The conformal coating of the copolymer film on an 8-mm hexagon head bolt was confirmed by scanning electron microscope (SEM)-energy dispersive Xray spectroscopy (EDS) analysis (Figure S3 in Supporting Information), as a conformal coating is required for antibacterial coatings of medical devices with various complex surface morphologies.


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Figure 2. (a) Surface atomic composition of quaternary ammonium (N+) (grey bar) and DMAEMA contents in each copolymer film (blue line). (b) Zeta potential and water contact angle of a series of p(VBC-co-DMAEMA), pV2D1, pV1D1, pV1D2, pV1D3.

2. Contact killing of bacteria on pV1D3 film To investigate the relationship between the copolymer film composition and antibacterial activities, the bacterial killing efficiency of the synthesized copolymer films was evaluated by using two types of bacteria, Escherichia coli (E. coli, a gram-negative bacterium) and Corynebacterium glutamicum (C. glutamicum, a gram-positive bacterium) (Figure 3a). With the increase in the quaternary ammonium proportion from pV2D1 to pV1D3, the antibacterial efficiency was increased against both E. coli and C. glutamicum. A dramatic enhancement in the


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bacterial killing activity was observed from pV2D1 to pV1D1 for both E. coli and C. glutamicum. The higher amount (~2-fold) of quaternary ammonium groups in pV1D1 than in pV2D1 was attributed to the excellent bacterial killing efficiency. Complete bacterial killing was achieved by pV1D3 against both E. coli and C. glutamicum. Interestingly, pV1D2 exhibited the highest quaternary ammonium fraction, whereas the best antibacterial performance was observed for pV1D3. This finding can be explained by the fact that antibacterial efficiency is related not only to the quaternary ammonium content but also to the surface wettability.32-33 The enhanced wettability of pV1D3 may overcome the lower amount of quaternary ammonium species. It is believed that more hydrophilic surfaces facilitate the initial interaction of bacteria with the surface, and thereby, the contact of bacteria with quaternary ammonium species is increased on pV1D3. Thus, the balance between the amount of quaternary ammonium species and surface wettability likely contributed to the superior performance of pV1D3 for killing bacteria. The bacterial killing efficacy over an extended time period was analyzed by counting the number of colonies (Figure 3b). Two bacterial types, E. coli and C. glutamicum, were incubated on pV1D3-coated plates for up to 168 h, and then the bacteria solution collected at specific time points was subjected to colony counting. Short-term incubation (1 h) of the bacteria showed 97% antibacterial efficiency. Complete bacterial killing effects were observed after 2 h of incubation. The plates that were exposed for more than 2 h maintained highly efficient antibacterial activity for up to 168 h with a minimal loss of antibacterial activity. In addition, we examined leaching of the pV1D3 films by NMR analysis after exposure to a mixture of D2O and PBS buffer (1:1, v/v) at 37°C for up to 5 days. No significant monomer peak corresponding to pV1D3 was observed (Figure S4 in Supporting Information). Both results indicated that the copolymer film possesses superior long-term antibacterial activity without any delamination or solubility


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problems in aqueous environments. These findings are explained by the fact that the crosslinking dramatically enhanced the stability of the copolymer film in aqueous medium by suppressing the leaching of antibacterial ionic functionalities from the surfaces, thus leading to remarkable antibacterial activity. To investigate the applicability of the antibacterial coating on a wide range of medical devices, pV1D3 was deposited on various substrate materials that are used to fabricate biomedical devices, and their antibacterial efficiencies on the surfaces were examined. Polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), and titanium were selected as target substrates because of their widespread use in medical devices. As shown in Figure 3c, the pV1D3-coated PET, PDMS, and Ti surfaces exhibited greater than 99.5% antibacterial efficiency against both E. coli and C. glutamicum. These results highlight that pV1D3 film deposition via iCVD is applicable for the modification of various substrates and is a promising approach for fabricating highly efficient antibacterial coatings for diverse biomedical devices.

Figure 3. (a) Antibacterial efficiency of each copolymer film against Escherichia coli (E.coli, a gram-negative bacteria) and Corynebacterium glutamicum (C.glutamicum, a gram-positive bacteria). (b) Antibacterial efficiency at different time point. Each bacteria cell was incubated in pV1D3-coated TCPS plate for those periods. (c) Antibacterial efficiency of pV1D3-coated PET, PDMS, and Ti. Each bacteria cell was incubated in each substrate for 2 h. The antibacterial efficiency was analyzed using colony counting assay.


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We next performed a FACS analysis to elucidate the impact of pV1D3 film on bacterial membrane disruption. As previously reported, two fluorescent dyes, SYBR Green I and propidium iodide (PI), were used to discriminate between intact and damaged bacterial membranes as part of a flow cytometry analysis34. The bacterial cells were incubated in pV1D3 film-coated plates and doubled-stained with PI and SYBR Green I dye. The majority of the population of bacterial cells exposed to pV1D3 film exhibited red fluorescence (Figure 4), indicating that the bacterial membrane for both bacterial strains was significantly damaged by contact with pV1D3 film. The population of membrane-compromised bacteria on the pV1D3 film was comparable to the positive control, ∼99% of E. coli and ∼97% of C. glutamicum bacteria killed. This observation suggested that the antibacterial mode of action of the pV1D3 film is associated with disruption of bacterial membranes by direct contact with quaternary ammonium species on the surface of pV1D3. This finding is in accordance with the generally proposed mode of action of quaternary ammonium compounds. The negatively charged bacterial membrane is susceptible to binding to the positively charged quaternary ammonium species, replacing Ca2+ and Mg2+ ions from the membrane, which leads to destabilization of the bacterial membrane and causes bacterial cell death.35


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Figure 4. Flow cytometry dot blots of (a) E.coli, (b) C.glutamicum. Each bacteria was incubated in pV1D3-coated plate and stained with SYBR Green I and propidium iodide dye.

3. Biocompatible pV1D3 film with highly efficient antibacterial capacity Inspired by the “race for the surface” concept, a co-culture study was designed and performed by following a previously established method with some modification.17 Co-cultures provide a powerful way to evaluate the responses of bacteria and mammalian cells that could occur in vivo on the implant surface. In this study, the pV1D3-modified surface was developed to kill bacteria prior to attachment of mammalian cells, which can efficiently prevent the formation of unfavorable environments that result from the rapid proliferation of bacteria (e.g., nutrient


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depletion, change in pH, and accumulation of toxins).36 The pV1D3-modified surface thereby provides a more favorable environment for the attachment and proliferation of mammalian cells. The biocompatibility of the pV1D3 film was evaluated using two mammalian cell types, murine fibroblasts (NIH-3T3) and human mesenchymal stem cells (hMSCs). The adhesion and spreading morphology of the cells grown on pV1D3-coated surfaces and control tissue culture plates (TCPS) were compared via microscopy after 24 h of culture. The microscopy images revealed that the pV1D3-coated surface supported normal cell adhesion and growth, and the cells exhibited similar cellular morphology and growth as those grown on the TCPS (Figure S5 in Supporting Information). The viability and proliferation of cells were evaluated by live/dead staining and a WST-1 assay after 24 h of culture. As shown in Figure 5a, all cells stained green on both the TCPS and the pV1D3-coated surfaces, implying that the pV1D3 film did not cause any cytotoxity. The WST-1 assay showed that the proliferation rates of NIH-3T3 cells and hMSCs on the pV1D3-coated surface reached 6.5±4.2% and 90.0±13%, respectively (Figure 5b), which were comparable to the rates observed on the TCPS. This result was consistent with that of the live/dead staining analysis, indicating that the pV1D3-coated surface supported the healthy growth and normal proliferation of mammalian cells without any inherent materialinduced toxicity. In addition, osteogenic differentiation of hMSCs was induced on the pV1D3coated surface, and the differentiated cells were examined by red S staining (Figure S6 in Supporting Information). Microscopy images revealed that the osteogenic differentiation capacity of hMSCs on the pV1D3-coated surface was comparable to that of TCPS-cultured cells after 21 days. These observations suggest that the pV1D3-coated surface exhibited properties that support mammalian cell growth and, thus, did not disturb the specific biological functions of the mammalian cells.


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Figure 5. Mammalian cell viability. Mouse fibroblasts (NIH-3T3) and human mesenchymal stem cells (hMSC) were cultured on tissue culture plates (TCPS) and pV1D3-coated surface. (a) Fluorescent images of live/dead assay on TCPS and pV1D3-coated surface after 24 h; Scale bar = 100 µm. (b) WST-1 assay of mammalian cells on TCPS and pV1D3-coated surface; Scale bar = 100 µm.

Next, we investigated the selective bacterial killing capability of the pV1D3 film in the presence of mammalian cells. It would be highly desirable for the copolymer film to retain antibacterial properties without damaging the surrounding tissue cells. Co-culture experiments with hMSCs were performed in a similar fashion to the previously reported method

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investigate the competition between mammalian cells and bacteria (C. glutamicum, 4.2×104 cfu/mL) for the pV1D3-coated surface. The viability of hMSCs after treating with a bacterial solution was analyzed by staining cells with FITC-phalloidin for F-actin (green) and DAPI for nuclei (blue) (Figure 6a).37-38 The fluorescent images revealed that the hMSCs on the pV1D3coated surface remained healthy with greater than 90% confluency after 24 h. By contrast, cells growing on TCPS were unable to adhere to the surface and, thus, only a few cells remained after 24 hr. The number of viable cells was calculated by staining the healthy cells (green) on both


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surfaces (Figure 6b). The cell growth rate of hMSCs on TCPS was significantly reduced relative to that of hMSCs on the pV1D3 film in the presence of bacteria. The number of healthy hMSCs on the pV1D3-coated surface gradually increased to up to 310 cells/field without any negative effect induced by the bacteria. However, the spreading of cells on TCPS was highly restricted, and only a small portion of cells (20 cells/field) remained after 24 h. Microscopy images also clearly showed the growth of bacterial cells on TCPS, which is the primary reason for the low survival rate of NIH-3T3 cells. In comparison, no bacterial cells were observed on the pV1D3coated plates (Figure 7). This result highlights the selective antibacterial characteristics of the pV1D3 film in efficiently killing bacteria prior to the adherence of mammalian cells to the surface. Thus, this approach can be considered as an innovative strategy for inhibiting the initial step of deep implant infections while promoting survival of the host cells. Considering that a low-dose inoculum, such as 100 colony-forming units (cfu) of bacteria, can infect 95% of an implant,39-40 the co-culture results show that surface modification with pV1D3 film is highly promising for preventing bacterial

Figure 6. Co-culture of bacteria and hMSC. (a) Fluorescent images of FITC-phalloidin stained F-actin (green), and DAPI stained nuclei (blue) of hMSC cultured on TCPS and pV1D3-coated surface. Fluorescent images were obtained after 6 h, 12 h, and 24 h of co-culture; Scale bar = 100µm. (b) The number of viable hMSC during 24 h of co-culture. ACS Paragon Plus Environment

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infection of implants.

Figure 7. The photographs and microscopy images showing co-culture of NIH-3T3 and E.coli (a), C.glutamicum (b) on TCPS and pV1D3-coated surface; Scale bar: 100µm. The survived bacteria cells were marked in red circles. We speculate that the selectivity of pV1D3 for bacteria over mammalian cells is associated with the combination of the quaternary ammonium species and the moderate wettability of the pV1D3 film. The selective bacterial killing by the quaternary ammonium species in pV1D3 may be due to the differences in the membrane structure and composition between mammalian cells and bacteria,41 by which the negatively charged bacterial membrane is able to bind to the quaternary ammonium species on the surface of pV1D3 and facilitate bacterial cell death. The intermediate hydrophilic properties of pV1D3 allow favorable initial interactions of the surface with both bacterial and mammalian cells, which increases the probability that bacteria contact the quaternary ammonium species on the pV1D3 surface, thus facilitating the contact killing of the bacteria. Conversely, the moderate wettability of pV1D3 encourages the attachment and survival


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of mammalian cells because mammalian cells adhere well to the surface, with contact angles of 40° - 60°.42

CONCLUSION In conclusion, we developed a highly robust antibacterial surface through the deposition of crosslinked ionic polymers, which yielded antibacterial surface coatings that selectively favor mammalian cells while killing bacteria. The antibacterial surfaces were generated via a one-step iCVD process, which allows in situ crosslinking during surface polymerization. The resulting quaternary ammonium groups on the surface of the copolymer network induced contact killing of bacteria. The pV1D3-modified surface maintained its antibacterial property for up to 168 h without leaching its key functional groups, implying that the crosslinking reaction enhanced the physicochemical stability of the copolymer film. The dependency of the antibacterial efficiency on the amount of surface quaternary ammonium groups on the copolymer film was investigated by examining the number of colony-forming bacterial cells (E. coli and C. glutamicum) that survived on the copolymer-coated surfaces. Among the copolymer films synthesized in this study, the pV1D3 film exhibited the highest bacteria-killing ability. FACS analysis suggested that the pV1D3 film functions by disrupting the bacterial membrane. A pV1D3-based antibacterial coating could be deposited onto various types of biomedical substrates such as PET, PDMS, and titanium while maintaining highly efficient antibacterial activity. According to the co-culture results, the pV1D3-coated surface provided a favorable environment for the attachment of mammalian cells while disrupting bacterial membranes. This selective killing of bacteria over mammalian cells will be highly beneficial for medical devices for clinical applications. The antibacterial coating method demonstrated in this work satisfies many of the


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essential criteria for medical applications, including strong antibacterial efficiency, compatibility with host cells, non-leaching properties, long-term efficiency, and compatibility with various types of substrate materials. We believe that the approach described here has great potential for the development of clinically applicable antibacterial surfaces.

EXPERIMENTAL SECTION 1. Materials 4-vinylbenzyl chloride (VBC, 90%), 2-(dimethylamino)ethyl methacrylate (DMAEMA, 95%), and tert-butyl peroxide (TBPO, 98%) were purchased from Sigma-Aldrich and were used without further purification. To analyze the properties of the polymeric thin films, the films were deposited onto glass slides (Marienfeld, Inc.) or Si wafers (Siltron, Inc.).

2. Synthesis of p(VBC-co-DMAEMA) copolymer films via iCVD To synthesize p(VBC-co-DMAEMA), the target substrate was inserted into the iCVD chamber (Daeki Hi-Tech, Inc.), and the temperature of the substrate was kept at 38 °C for the uniform adsorption of monomers during iCVD. DMAEMA and VBC monomers and the TBPO initiator were then introduced into the iCVD chamber to synthesize the copolymer (p(VBC-coDMAEMA)). To vaporize each reactant, VBC and DMAEMA were heated to 60 °C and 35 °C, respectively, and the initiator was vaporized at room temperature. To fabricate a series of copolymer films, the flow ratio of each monomer, VBC and DMAEMA, was adjusted by the needle valve. For the pV2D1 film, the feed rates of VBC, DMAEMA and TBPO were maintained at 1.4, 0.7 and 0.8 sccm. For the other copolymer thin films, the feed rate of TBPO


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was fixed at 0.8 sccm. The detailed feed ratios for each copolymer are summarized in Table S1. Simultaneously, the inner chamber pressure was kept at 200 mTorr, and then the temperature of the filament was heated to 180 °C to make TBPO radicals for the free radical polymerization reactions. The film thickness was monitored in situ by a He-Ne laser (JDS Uniphase) interferometer.

3. Polymer film characterization Fourier-Transform Infrared spectroscopy results were tracked using the ALPHA FT-IR (Bruker Optics) instrument in absorbance mode, with 64 scans collected for each spectrum to confirm the copolymerization between VBC and DMAEMA. The deposition of copolymer films in the iCVD chamber was analyzed by X-ray Photoelectron Spectroscopy, or XPS (Sigma Probe, Thermo VG Scientific, Inc.) measurement. From the XPS data, the ratio of atoms corresponding to ionic components (N+, Cl-) was calculated by using peak deconvolutions. The cross-linked ratio of the copolymers was calculated from the ionic contents by dividing the atomic percent of N1s + by the sum of the atomic percent of the elements that participate in the crosslinking reaction and the unreacted elements [N1s +, Cl2p + (N1s + or Cl2p −)]. To ensure the p(VBC-co-DMAEMA) deposition on the

substrates after the iCVD process, the static water contact angles (WCAs) were measured by a contact angle analyzer (Phoenix 150, SEO, Inc.). The volume of individual water droplets was 10 µL. The zeta potential of each copolymer film-coated glass substrate was measured using a zeta potential analyzer (ELSZ-1000ZS, Otsuka Electronics). The coated glass substrates were cut to a size of 10 mm × 30 mm and installed onto the flat surface cell unit for measurement. The monitor particle was dispersed in NaCl solution (10 mM). After the NaCl solution was injected into the cell, the zeta potential was measured by flowing the dispersed monitor particle into the flat zeta cell unit. Atomic force microscope (AFM) images of the polymer films were collected


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with scan sizes of 2 µm × 2 µm using a scanning probe microscope (XE-100, Park Systems). Scanning electron microscopy and energy disperse spectroscopy (SEM-EDS) (Magellan 400, FEI Company) were used to analyze the surface morphology and surface chemical composition of pV1D3-coated 8 mm hexagon head bolts. Nuclear magnetic resonance, or NMR analysis (Agilent 400 MHz 54 mm NMR DD2, Agilent Technologies) was used to determine the leaching of the copolymer films. The pV1D3-coated TCPS was incubated in a D2O/PBS buffer mixture (1 mL), followed by 1H-NMR analysis at 1, 3, and 5 days.

4. Antibacterial efficiency The antibacterial properties were analyzed according to ASTM E2149-01. E. coli XL1-Blue (Stratagene, USA) and C. glutamicum ATCC13032 (American Type Culture Collection, Manassas, VA, USA) cells were inoculated into Luria-Bertani (LB) media (5 mL) and incubated at 37 °C and 30 °C, respectively. After overnight cultivation, the cells (50 µL) were transferred into fresh LB media (5 mL) and cultured until the optical density at a wavelength of 600 nm reached 0.6. The cells were diluted in sodium phosphate buffer (10 mM, pH 7.4) to a concentration of 104 cells/mL. The cell suspension (1 mL) was incubated in copolymer filmdeposited plates at room temperature for 2 h, followed by spreading on agar plates for a colony counting assay. For bacterial killing efficacy over an extended time period, copolymer filmdeposited plates were exposed to bacteria cell suspension for up to 168 h, and then the cell suspension at various time points were collected for colony counts. The antibacterial efficiencies were quantified by counting the colonies of reacted cells with antibacterial films. Positive control samples were prepared by treating the cells with 50% (v/v) isopropanol for 2 h. For the colony counting analysis, E. coli and C. glutamicum cell suspensions (100 µL) were spread on 1% agar-


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LB plates, and the numbers of colonies were counted after overnight incubation. For the flow cytometry analysis, the collected cell suspensions were incubated with PI (10 µM, SigmaAldrich) and SYBR Green I (Sigma-Aldrich). After 5 min of incubation, the fluorescence signals of the stained cells were analyzed using flow cytometry (MoFlo XDP, Beckman Coulter, USA). The fluorescent intensities of the cells were measured at a wavelength of 525 nm for SYBR Green I and 620 nm for PI.

5. Mammalian cell viability Mouse NIH-3T3 fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco) in an atmosphere of 5% CO2 at 37 °C. hMSCs were cultured in minimum essential medium alpha (MEM Alpha, Gibco) supplemented with 17% FBS and 1% penicillin/streptomycin in a 5% CO2 atmosphere at 37 °C. For the cell viability test, 3×105 NIH-3T3 cells and hMSCs were seeded on 35-mm TCPS (SPL) and pV1D3-coated 35-mm TCPS. The cells were cultured for 1 day, washed with Dulbecco’s phosphate-buffered saline (DPBS), and stained with a LIVE/DEAD® viability/cytotoxicity kit for mammalian cells (Thermo Fisher Scientific). Each sample was incubated with DPBS (1 mL) containing ethidium homodimer-1 (4 µΜ) and calcein (2 µΜ) for 45 min at room temperature. All samples were subsequently rinsed with DPBS and observed under a fluorescence microscope (Eclipse Ti-U, Nikon). For the cell proliferation assay, 104 NIH-3T3 cells and 103 hMSCs were seeded and cultured on 96-well plates and pV1D3-coated 96-well plates for 24 h. After each well was washed with DPBS, growth media containing 10% WST-1 reagent (Roche) was added to each well, and the cells were incubated for 3 h at 37 °C


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with 5% CO2. The absorbance of the samples was measured using a microplate reader (Gemini XPS Microplate Reader) at 450 nm. The reference wavelength was 690 nm.

6. Alizarin red S staining hMSCs at a concentration of 3×105 were cultured for 21 days on the 35-mm TCPS and pV1D3coated TCPS with hMSC osteogenic differentiation media (hMSC Osteogenic BulletKit, Lonza). The cells were then fixed with 4% paraformaldehyde for 15 min at room temperature, washed with ultra-pure water (Gibco), and soaked with Alizarin Red S staining solution (1 mL, ScienCell) for 25 min at room temperature. After the cells were washed with ultra-pure water, the stained cells were imaged by microscopy.

7. Bacterial & mammalian cell co-culture In vitro bacteria and cell co-culture experiments were performed according to Subbiahdoss and co-workers, with some modifications.17,36 A 1-mL volume of bacterial suspension (C. glutamicum) (4.2×104 cfu/mL) was distributed over the TCPS and pV1D3-coated TCPS and incubated at 37 °C for 2 h. After washing the plates with DPBS, 3×105 hMSCs were seeded on the bacteria-contaminated plates with a modified medium consisting of 98% growth medium and 2% LB broth and cultured in a humidified 5% CO2 atmosphere at 37 °C for 1 day. After 6 h, 12 h, and 24 h, the samples were fixed with 4% paraformaldehyde and stained with FITC-phalloidin (Thermo Fisher Scientific) and DAPI (Sigma-Aldrich). Each plate was examined by fluorescence microscopy, and the numbers of viable cells were calculated by using ImageJ software.

ASSOCIATED CONTENT


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Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. XPS analysis, water contact angle, AFM images, SEM-EDS analysis, 1H-NMR analysis, microscope images, Alizarin Red S staining.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (K.J.) *E-mail: [email protected] (E.L.) Present Addresses †R&D Center, GS Caltex Corporation, Daejeon 34122, Republic of Korea. (G.J.) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This study was supported by the Wearable Platform Materials Technology Center (WMC) funded by the National Research Foundation of Korea (NRF) Grant of the Korean Government


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(MSIP) (No. 2016R1A5A1009926), and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2017R1A2B3007806).

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For Table of Contents Use Only

A robust thin film surface with a selective antibacterial property enabled via a cross-linked ionic polymer coating for infection-resistant medical applications Goro Choi‡, Gu Min Jeong‡, Myung Seok Oh‡, Munkyu Joo, Sung Gap Im, Ki Jun Jeong*, Eunjung Lee* Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Daejeon 34141, Republic of Korea. *

Corresponding authors: [email protected], [email protected].


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A robust thin film surface with a selective antibacterial property

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