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Non-leachable imidazolium-incorporated composite for disruption of bacterial clustering, exopolysaccharide matrix assembly and enhanced biofilm removal Geelsu Hwang, Bernard Koltisko, Xiaoming Jin, and Hyun Koo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11558 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017

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ACS Applied Materials & Interfaces

Non-leachable imidazolium-incorporated composite for disruption of bacterial clustering, exopolysaccharide matrix assembly and enhanced biofilm removal

Geelsu Hwang1, Bernard Koltisko2, Xiaoming Jin2,*, Hyun Koo1,*

1

Biofilm Research Labs, Levy Center for Oral Health, Department of Orthodontics and

Divisions of Pediatric Dentistry & Community Oral Health, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

2

Dentsply Sirona, Milford, Delaware 19963, USA

* Corresponding authors

Xiaoming Jin, 38 West Clarke Ave., Milford, DE 19963; Tel: (302) 430-7407; E-mail: [email protected];

Hyun Koo, 240 South 40th Street, Levy Bldg. Rm 417, Philadelphia, PA 19104-6030; Tel: (215) 898-8993; E-mail: [email protected]

Key words: antibiofilm, imidazolium-containing resin, dental composite, Streptococcus mutans, biofilms, EPS-matrix, mechanical stability 1

ACS Paragon Plus Environment


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Abstract. Surface-grown bacteria and production of an extracellular polymeric matrix modulate the assembly of highly cohesive and firmly attached biofilms, making them difficult to remove from solid surfaces. Inhibition of cell growth and inactivation of matrixproducing bacteria can impair biofilm formation and facilitate removal. Here, we developed a novel non-leachable antibacterial composite with potent antibiofilm activity by directly incorporating polymerizable imidazolium-containing resin (Anti Bacterial Resin with Carbonate linkage; ABR-C) into a methacrylate-based scaffold (ABR Modified Composite; ABR-MC) using an efficient yet simplified chemistry. Low-dose inclusion of imidazolium moiety (~2% wt/wt) resulted in bioactivity with minimal cytotoxicity without compromising mechanical integrity of the restorative material. The antibiofilm properties of ABR-MC were assessed using an exopolysaccharides (EPS)-matrix producing oral pathogen (Streptococcus mutans) in an experimental biofilm model. Using high-resolution confocal fluorescence imaging and biophysical methods, we observed remarkable disruption of bacterial accumulation and defective 3D matrix structure on the surface of ABR-MC. Specifically, the antibacterial composite impaired the ability of S. mutans to form organized bacterial clusters on the surface, resulting in altered biofilm architecture with sparse cell accumulation and reduced amounts of EPS-matrix (vs. control composite). Biofilm topology analyses on the control composite revealed a highly organized and web-like EPS structure that tethers the bacterial clusters to each other and to the surface, forming a highly cohesive unit. In contrast, 2


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such structured matrix was absent on the surface of ABR-MC with mostly sparse and amorphous EPS, indicating disruption in the biofilm physical stability. Consistent with lack of structural organization, the defective biofilm on the surface of ABR-MC was readily detached when subjected to low shear stress, while most of the biofilm biomass remained on the control surface. Altogether, we demonstrate a new non-leachable antibacterial composite with excellent antibiofilm activity without affecting its mechanical properties, which may serve as a platform for development of alternative anti-fouling biomaterials.

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Introduction Biofilms are comprised of dense, highly hydrated clusters of microbial cells that are embedded in an extracellular matrix of polymeric substances, such as exopolysaccharides (EPS), proteins, and nucleic acids 1,2. In particular, EPS-matrix enhances cell adhesion and cohesion that promote both microbial accumulation onto a surface and the development of densely packed cell aggregates, resulting in a highly structured and adherent biofilm 2-4. Moreover, bacteria embedded in the protective matrix are up to 1,000 times more resistant to antibiotics than bacteria in planktonic phase 5. Thus, once biofilms are established, it becomes extremely difficult to kill or mechanically remove from solid surfaces. Contamination of implants and other medical devices by biofilms can lead to severe and costly secondary infections 6. Biofilm formation on restorative and dental implant materials is commonly associated with clinical failure, and the onset of various oral diseases such as tooth-decay and peri-implantitis 7,8. Therefore, inhibition of microbial adhesion or inactivation of the adhered bacteria could impair their development into biofilms.

Development of effective antibiofilm materials is challenging three-fold; it should provide effective antimicrobial activity and EPS inhibition in situ, superior biocompatibility, and optimal physico-mechanical properties. Extensive efforts have been made to engineer new antibacterial surfaces or improve the performance of existing surfaces by applying surface coatings or modifying surface chemistry 8-13. For example, several approaches to inhibit 4


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bacterial adhesion and growth have been developed by incorporating nitric oxide-releasing particles 14-16, metallic alloy elements 17-19, proteins 20,21, and nanoparticle-polymer hybrids 22,23

into a variety of substrates. In particular, restorative dental materials have been actively

explored for incorporation of biologically active components such as antimicrobials, resulting in composites exhibiting slow-release of biocides 16,24-28. However, antimicrobial reservoirs are often subject to progressive decreases in efficacy (short-term effect) through gradual drug release into the surrounding environment, which could also lead to development of antibacterial resistance 29. Alternatively, polymerizable quaternary ammonium salt (QAS) resins or nanoparticles based on polymeric QAS can be covalently immobilized, leading to non-leaching antimicrobial surfaces 30-36. Despite of conceivable advantage of non-leaching materials for long-lasting antimicrobial effect, conventional QAS-based resin often requires high-dose drug loading (up to 30%), thereby compromising mechanical durability and increasing cytotoxicity of surfaces 37. Furthermore, effectiveness of QAS-nanoparticles incorporated resin varied as their antibacterial activity depends on particle size38 and 3D nanoscale surface roughness 39. Thus, there is a need for development of new and effective non-leaching antibacterial restorative materials without compromising its mechanical integrity.

Recently, an improved and simplified method of producing polymerizable antibacterial resins has been developed 40. Here, we devised a novel imidazolium-containing polymerizable resin 5



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(3-dodecanylimidazoliumbromide dimethacrylate resin, ABR-C) and corresponding antibacterial composite (ABR-MC) formulated from ABR-C. Low dose loading of the bioactive and chemically polar ABR-C (~2% wt/wt) allowed the formulation of ABR-MC containing non-leachable imidazolium moieties without negatively impacting its mechanical properties. In turn, the experimental composite provided excellent antibacterial and biofilm disruptive properties. Our data reveal strong inhibition of bacterial growth that impaired the formation of structured cell clusters or aggregates on the surface of ABR-MC, resulting in severely defective biofilms containing mostly amorphous and disorganized EPS. Conversely, biofilms formed on the control composite harbored large cell clusters tethered into a highly organized and web-like EPS structure. Consistent with such structurally cohesive scaffold, most of the biofilm biomass remained on the control surface when subjected to high shear forces (1.78 N/m2). In contrast, the defective biofilm was readily detached from ABR-MC surface even when low shear stress (0.18 N/m2) was applied. Importantly, the anti-biofilm efficacy of re-used ABR-MC, after cleaning (with brushing followed by 10 min of bath sonication) and autoclaving, was maintained. Together, the antibiofilm properties combined with simplified chemical synthesis of this imidazolium containing antibacterial composite may serve as a platform to further develop restorative dental materials and other non-leaching anti-fouling surfaces.

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Experimental Section Synthesis of Polymerizable Antibacterial Resin, ABR-C. The synthesis process of ABR-C containing imidazolium moiety (a well-known antimicrobial compound 41) is described in Figure 1. Briefly, a monoimidazol-containing dimethacrylate was prepared via the reaction of 3-(acryloyloxy)-2-hydroxypropyl methacrylate (AMAHP) and 1,1'-carbonyldiimidazole (CDI), which was then directly coupled with 2-Hydroxyethyl methacrylate (HEMA) to yield a monoimidazole-dimethacrylate resin (precursor to ABR-C). The precursor resin was then reacted with 1-bromododecane (C12Br) to convert the imidazole moiety into imidazolium moiety as ABR-C. The details of the chemical synthesis are described as follows.

In step 1, 170.3 g (1.05 mol) of 1,1'-Carbonyldiimidazole (CDI), 380 ml of methylene dichloride and 215.4 g (1.005 mol) of 3-(acryloyloxy)-2-hydroxypropyl methacrylate (AMAHP) was charged into a 1000 ml 3-neck round flask, and allowed to react for 6 hr at room temperature. Subsequently, 135.0 g (1.037 mol) of HEMA, 40 g of potassium carbonate and 4.0 g of tetra-n-butylammonium bromide (tBAB) were added, and allowed to react for an additional 10-12 hrs at room temperature, followed by adding 200 ml of DI water to stop the reaction (Step 2). The resulting solution was extracted 4-5 times with DI water to remove all of the unreacted imidazole. Then, the solution was dried over magnesium sulfate overnight at room temperature prior to being filtrated. The solvent was removed via Rotovapor at 35-40 °C under vacuum and low-viscous (3.0 Pa·s at 20 °C) liquid resin is resulted (421g as 96 % in 7



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yield). This resin was further characterized by 1H NMR (Figure S1A) and 13C-NMR (Figure S1B). It is noteworthy that an alternative approach to a variety of monoimidazolemonmethacrylate resins was also developed by using imidazole as illustrated in Figure S2. In step 3, the prepared precursor resin was reacted with 1-bromododecane (C12Br) to convert the imidazole moiety into imidazolium moiety; 350.9 g of precursor resin (as described above) and 205.7 g of 1-bromododecane (C12Br) were charged into a 1000 ml resin kettle, being reacted for 8 days at 40-45 °C. The resulting liquid resin was dissolved in 300 ml methylene dichloride and precipitated in hexane. Then, it was further dried under vacuum and 483 g of liquid resin is collected in yield of 88 % (175.0 Pa·s at 20 °C). This resin was further characterized by 1H NMR (Figure S1C) and 13C-NMR (Figure S1D), respectively. Assignment of key protons and carbons for NMR is listed in the Supporting Information.

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Figure 1. Schematic diagram of two-pot three-step synthetic route to 3dodecanylimidazoliumbromide dimethacrylate carbonate (ABR-C). Precursor to ABR-C was prepared via one-pot two-step reaction (Step 1 and 2), and ABR-C was prepared via additional one-pot reaction (Step 3). The circle in Step 3 indicates the incorporated imidazolium moiety into ABR-C.

General Manufacturing Procedures of Composites Based on ABR-C. In addition to ABRC, other conventional resins, initiators/inhibitor and glass fillers used in formulations were included as follows: 1,6-Bis [methacryloyloxyethoxycarbonylamino]-2,4,4- trimethylhexane 9



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(UDMA); EBPADMA; a urethane-modified BisGMA dimethacrylated resin (TPH Resin); Camphorquinone (CQ); Diphenyl (2, 4, 6-trimethylbenzoyl) phosphine oxide (L-TPO); Dimethylaminobenzonitrile (DMABN); Butylated Hydroxytoluene (BHT); barium fluoroalumino borosilicate glass surface treated by γ- methacryloxypropyl-trimethoxysilane (Silanated BFBG-1; silanated As Rec’d BaBFAlSiO4, 4-7 μm of mean particle size); barium fluoroalumino borosilicate glass surface treated by γ- methacryloxypropyltrimethoxysilane (Silanated BFBG-2; silanated Milled Ultra Fine BaBFAlSiO4, 0.92-0.96 μm of mean particle size); Silanated Strontium-AluminoSodium-Fluoro-Phosphorsilicate glass surface treated by γ-methacryloxypropyl-trimethoxysilane (Silanated SAFG; silanated OX-50, 50 nm SiO2). Dental composite containing polymerizable imidazolium resin (ABR-C at 2.1 % wt/wt) was compounded with 77 % of filler mix by using a Ross mixer at 50 °C under reduced pressure. The inorganic fillers used here is blend of three types of particles: 28.6% wt/wt of BFBG1, 57.2% wt/wt of BFBG2, and 14.2% wt/wt of SAFG.

Measurement of Mechanical Properties. Specimens for 3-point bending flexural test were prepared according to ISO 4049 42. Composite was filled into 25 mm × 2 mm × 2 mm stainless steel mold, then covered with Mylar film and cured using Spectrum 800 (DENTSPLY Caulk) halogen lamp at intensity of 550 mW/cm2 for 4 × 20 s uniformly across the entire length of the specimen. The set specimens were stored in deionized water at 37°C 10


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for 24 hours prior to the test. Flexural test was conducted using an Instron Universal Tester (Model 4400R, Instron Corp., Canton, MA) with crosshead speed 0.75 mm/min under compressive loading mode. A minimum of six specimens were tested for each set of sample. Compressive strength and modulus were measured by filling the composite into Ø 4 × 7 mm Teflon molds and sandwiched between two Mylar cover films, then cured using Spectrum 800 lamp at intensity of 550 mW/cm2 on both ends. The set specimens were stored in deionized water at 37°C for 24 hours prior being polished to 6 mm long x 4mm in diameter using 600 grit sandpaper. Compression test was conducted using an Instron Universal Tester Model 4400R with crosshead speed 5 mm/min. Six specimens were tested for each set of sample.

Bacterial Strain. Staphylococcus aureus ATCC 6538 was used to screen the antibacterial activity on all formulated compositions with variable ABR-C contents, according to ISO 22196: 2011 43. Streptococcus mutans UA159 (ATCC 700610), a virulent cariogenic pathogen and well-characterized EPS-matrix producing, biofilm forming and acidogenic/aciduric strain 44, was used to test antibacterial activity, determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as well as assess antibiofilm effects of ABR-MC. The cultures were stored at – 80 °C in tryptic soy broth containing 20 % glycerol. 11



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Antibacterial Test. Composite was filled into 2” × 2” × 2 mm stainless steel mold, then covered with Mylar film and cured using Triad 2000 (DENTSPLY Caulk) for 2 min uniformly on each side. No further polish on cured composite prior to the test at Antimicrobial Test Laboratories (Texas) according to ISO-22196 test protocol 43, which is an industry standard method for antibacterial evaluation as detailed in Supporting Information. Briefly, test microorganism (S. aureus) is inoculated in the culture medium, and the bacterial suspension standardized by optical density and culturing as detailed in ISO-22196 protocol. Control composite and ABR-MC surfaces were inoculated with microorganisms, and then the microbial inoculum was covered with a thin, sterile film. The inoculum spreads were covered to prevent the evaporation and to ensure close contact with the antimicrobial surface. Microbial concentrations were determined at "time zero" by elution followed by dilution and plating to agar, and counting the colony forming units (cfu). A control was run to verify that the neutralization/elution method effectively neutralizes the antimicrobial agent in the antimicrobial surface being tested. Inoculated, covered control and ABR-MC surfaces were allowed to incubate undisturbed in a humid environment for 24 hr at 37 °C. Finally, microbial concentrations were determined. Reduction of microorganisms relative to the microbial concentration at time zero was calculated. All assays were done in triplicate in at least three different experiments.

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Antibiofilm Test. Composite was filled into Ø 1.5 × 10 mm Teflon molds, then covered with Mylar film and cured using Spectrum 800 (DENTSPLY Caulk) halogen lamp at intensity of 550 mW/cm2 for 20 s uniformly on each side. Then the cured specimen were further polished on a grinding wheel with two types of sanding paper of 600 and 1200 grid, respectively. S.

mutans UA159 was used to form biofilms on the surface of the test materials. Biofilms were grown on saliva-coated control and ABR-MC discs (surface area, 2.7 ± 0.2 cm2) vertically suspended in 24-well plates using a custom-made wire disc holder as detailed previously 44,45. Briefly, each disc coated with filter-sterilized, clarified whole saliva was vertically suspended in 24-well plates using a custom- disc holder, mimicking the free smooth surfaces of the pellicle-coated teeth made wire 44,46. Then, each disc was inoculated with ~2 × 102 CFU of S.

mutans ml−1 in ultrafiltered (10 kDa cut-off; Millipore, Billerica, MA, USA) yeast tryptone extract broth (UFTYE) containing 1% sucrose at 37°C and 5% CO2. The culture medium was then replaced twice daily (at 8 am and 6 pm) until the end of the experimental period (67 h), and the pH values measured daily using an Orion pH electrode attached to an Orion 290A+ pH meter (Thermo Fischer Scientific) . The biofilms were collected at different time-points, and analyzed for dry-weight and by means of fluorescence imaging using multi-photon confocal microscope SP5 (Leica Microsystems, Buffalo Grove, IL,USA) equipped with a 20 X (1.0 numerical aperture) water immersion lens. EPS were labelled via incorporation of Alexa Fluor 647 dextran conjugate (Molecular Probes Inc., Eugene, OR) during biofilm 13



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formation, while S. mutans cells were stained with Syto 9 (485/498 nm; Molecular Probes) as detailed previously 44 and in Supporting Information. Each biofilm was scanned at 5 positions randomly selected on the microscope stage, and confocal image series were generated by optical sectioning at each of these positions. Quantitative computational analyses via Image J and COMSTAT were conducted for measurement of EPS and bacterial biomass across intact 3D biofilm architecture as detailed previously 44,45. For testing re-used composites, we gently brushed with a soft-toothbrush and sonicated used composites (both control and ABR-MC) in water bath for 10 min to remove biomass. Then, the composites were autoclaved and the bioactivity evaluated as described above. All assays were done in triplicate in at least three different experiments.

Analysis of the Attachment Strength and Structural Integrity of Biofilms. The mechanical stability of biofilms on each composite (control and ABR-MC) surfaces was compared using a custom-built device 47, which is capable of generating a diverse range of shear stress to remove S. mutans biofilms from saliva-coated surfaces (see details in Supporting Information). Biofilms formed on each composite were placed in the disk holder of the device, and then exposed to constant shear stress of 0.18, 0.81, and 1.78 N/m2 for 10 min. Based on our previous study, the duration of 10 min of shearing was determined to have reached a steady state of biofilm removal 47. The amount of remained biofilm dry-weight 14


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(biomass) for each condition was determined before and after application of shear stress. The percentage of biofilm removed from the composite surfaces was calculated and also visualized using confocal microscopy as described previously 47. All assays were done in quadruplicate in at least three different experiments.

Statistical Analysis. Statistical analyses for the experimental data were performed using regression models to obtain overall tests of equality and pairwise comparisons. KruskalWallis tests, which are non-parametric and based on ranks, were used for two-group comparisons. The significance levels were set at 5 %.

Results Synthesis of Imidazolium-based Polymerizable Resin (ABR-C). Previously, we unexpectedly found that the by-product imidazole from the primary reaction between 1, 1’carbonyl-diimidazole (CDI) and the secondary OH in an acrylate-methacrylate hybrid resin (AMAHP) could simultaneously and exclusively react with the acrylate group 40. Hence, this ‘dual-reaction in one-pot’ via CDI could be used as facile process to develop a variety of imidazole-containing resins. Here, we report a polymerizable antibacterial resin (denoted as ABR-C; Figure 1), which structurally features bismethacrylate as the polymerizable group 15



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and imidazolium bromide as the charged moiety and dodecanyl (C12H25) as the alkyl group. We first prepared the imidazole-containing precursor to ABR-C in one-pot two-step process by using an AMAHP and CDI (Figure 1). Imidazole was covalently added to the acrylate double bound via Michael addition without any catalysts as the OH was activated as carbamoyl-imidazole, which readily reacted with HEMA to generate the second polymerizable group in the parent AMAHP core via carbonate linkage. The molecular structure of this precursor was elucidated by both 1H NMR and 13C NMR analysis (see Figure S1A, B for detailed assignments of the key chemical shifts). The precursor was then converted to ABR-C via chemical modification on the imidazole moiety to yield polymerizable resins with ionic moiety of imidazolium. This was accomplished by reacting the precursor directly with 1bromododecane without addition of catalysts as verified by NMR analysis on its molecular structure (Figure S1C, D). Collectively, we successfully prepared a polymerizable imidazolium-containing resin.

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Table 1. Compositions and properties of composites with and without antibacterial resin. Control

ABR-MC

(TPH* Resin)

(TPH Resin/ABR-C)

23.0 %

20.9 %

(0%)

(2.10 %)

Filler Blend, wt/wt

77.0 %

77.0 %

Compr. St. (MPa)

306 ± 50

313 ± 17

5160 ± 470

5630 ± 200

128 ± 14

111 ± 10

11760 ± 490

9180 ± 460

CFU at 0 hr

1.75x105

1.75x105

CFU at 24hr

2.10x106

non detected

Resin Blend, wt/wt (ABR-C, wt/wt)

Compr. Mod. (MPa) Flex. St. (MPa) Flex. Mod. (MPa) Antibacterial (ISO 22196)**

(Reduction, %)

>99.99

* TPH is a commercially available, conventional dental composite manufactured by Dentsply Sirona. ** Staphylococcus aureus ATCC 6538 was tested according to ISO 22196.

Antibacterial Activity and Mechanical Properties of ABR-MC. Since imidazolium moieties feature antimicrobial properties, we first examined whether the uncured polymerizable imidazolium-based resin is capable of bacterial killing (MIC and MBC; see Table S1). Then, we assessed the antibacterial activity of cured ABR-MC against Staphylococcus aureus ATCC 6538, according to ISO 22196: 2011 43. S. aureus was used since it is one of the 17



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standard strains for antibacterial testing according to ISO 22196 43. As expected, we observed almost complete killing of the bacterial cells (>99.99%) on the surface of ABR-MC despite incorporation of low amounts of ABR-C (~2%, wt/wt in total composition). We also confirmed S. mutans killing on the ABR-MC composite surface (Figure S2). Excitingly, there was no negative impact on the mechanical properties (flexural, compressive strength and modulus), which indicates strong resistance of ABR-MC to breaking under compression. Furthermore, cytotoxicity data from both ISO agarose overlay test and elution method show that ABR-MC has less than or equal to grade 2, which is considered negligible cytotoxicity against mammalian cells (L-929 mouse fibroblast cells) (Table S2). Altogether, we developed an ABR-modified composite (ABR-MC) using simplified chemistry that achieved an excellent balance between antibacterial activity and low cytotoxicity without compromising the mechanical integrity.

Antibiofilm Property of ABR-MC. The antibacterial properties of ABR-MC surface may lead to inhibition of biofilm assembly and further accumulation. In addition, our preliminary study showed that bacterial binding to ABR-MC surface was also disrupted (Figure S4). Thus, we examined the dynamics of S. mutans biofilm formation using our in vitro model 44-46. Biofilms were grown on control or ABR-MC, and biofilm formation was analyzed at various time points representing early, mid and late-stages of development under our experimental 18


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conditions (29, 43, and 67 h). Representative confocal images revealed structured bacterial clusters or microcolonies (in green) and the presence of an EPS rich-matrix (in red) in the biofilms formed on the control composite surface (Figure 2A). In sharp contrast, the biofilm initiation and accumulation as well as its structural organization were significantly compromised on ABR-MC showing small bacterial clusters with minimal EPS only at the later time point (67h). Indeed, biofilm biomass (dry-weight) accumulated onto the control composite over time, while negligible amounts were detected at 29 and 43h on ABR-MC, which agrees well with the confocal imaging data (Figure 2B-D). Although bacterial clusters are observed on ABR-MC at 67 h, they were sparsely distributed across the surface. Consistent with the imaging and biomass data, the culture pH values of the biofilms formed on the control composite steeply dropped, indicating active metabolic activity (i.e. acid production) as the biofilm accumulates on the surface (Figure 2E), and were significantly lower than those from ABR-MC (P40µm height; Figure S5). This lack of organization of the EPS-matrix could have weakened the cohesiveness and attachment 25



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strength of the defective biofilms formed on ABR-MC, and thereby facilitated biofilm removal when subjected to external shear stress. Collectively, our data reveal that ABR-MC can significantly disrupt both the biofilm initiation and its further development, while also hindering the formation of dense and structurally organized (web-like) EPS-matrix, an important feature of well-established and adherent biofilms.

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Figure 5. Representative projection images of skeletonized EPS-matrix from 67h biofilm formed on each composite surface. (A) Overall and (C) close-up views of skeletonized EPSmatrix on control surface, and (B) overall and (D) close-up views of skeletonized EPS-matrix on ABR-MC surface. White arrows indicate the ‘superstructure’ filled with a dense mesh of thin filaments crisscrossing the thicker EPS.

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Discussion Advances in surface chemistry and engineering have led to a range of biomaterials with multiple applications for human use, including, orthopedic, nasal and dental implants 50-52. Current modalities of antibacterial surface focus primarily on disrupting initial bacterial adhesion and colonization or killing to help control biofilm formation and the onset of implant-associated infections 53,54. Particularly, resin composites for dental restoration require these beneficial properties without compromising their mechanical integrity to minimize premature failure (e.g. micro-factures) and frequent replacements 26,27,55. However, antimicrobial surfaces in general have limitations to reduce further biofilm accumulation over-time as they are often incapable of disrupting the EPS matrix 56. Here, we successfully developed a novel polymerizable imidazolium-incorporated non-leachable antibacterial composite, which exhibited potent biofilm disruptive activities with minimal cytotoxicity and without negatively impacting the mechanical properties of the material. Furthermore, we demonstrated how the antibacterial surface could modulate the accumulation and physical integrity of biofilms by disrupting the formation and structural organization of the EPS matrix. To our knowledge, this work provides new insights about EPS matrix assembly on an antibacterial surface, which remain largely unexplored despite its recognized importance on biofilm development, surface attachment and antimicrobial tolerance 4,57.

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Organic compounds bearing imidazolium moieties have shown various biomedical properties such as antibacterial and antifungal activities 41,58,59. However, development of polymerizable resin containing imidazolium moiety could be challenging, as it should be designed to maintain both mechanical integrity and antimicrobial activity. In this study, we report an efficient yet facile method to incorporate an imidazole moiety into a polymerizable acrylatemethacrylate hybrid resin via a Michael addition without the need to include catalysts as shown in Figure 1. Consequently, such imidazole-containing polymerizable resin was readily converted into a monoimidazoliumbromide-dimethacrylate resin by a one-step reaction (as verified via 1H NMR and 13C NMR spectra; Figure S1C, D). In addition, an alternative process towards imidazole-containing polymerizable resin was also developed via direct imidazole Michael addition to AMAHP without use of CDI as illustrated in Figure S3. This simplified method can facilitate the production of a variety of new class polymerizable monoimidazole monomethacrylate resins, which can provide a wide range of opportunities to further optimize its properties and functionalities.

The mechanism of antimicrobial action of the imidazolium salts is similar to that of QAS, which disturbs the cell membrane, resulting in bacterial killing 41. Thus, we examined the bacterial viability and antibiofilm properties of ABR-MC against S. mutans, an established biofilm-forming and EPS-producing oral pathogen. The data revealed potent antibacterial activity, resulting in significantly impaired biofilm formation on the surface of ABR-MC, 29



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compared with control composite. The chain length of the N-alkyl group has been shown to be critical for antimicrobial efficacy of the imidazolium salt with optimal number of chains between 10 and 14 carbon atoms 60-63. ABR-MC is formulated to have linear alkyl chains with 12 carbon atoms (see Figure 1), which exhibited significant bacterial killing and effective disruption of biofilm initiation on ABR-MC. Furthermore, synthesis of nonleachable antibiofilm surface without chemically grafting antibiotics to the substratum may provide long-lasting effects 64. We observed that the bioactivity of reused ABR-MC (after cleaning and autoclaving) was mostly maintained (Figure 2D), indicating (albeit indirectly) that the majority of imidazole-containing monomer is anchored into the polymer network. However, its efficacy was slightly reduced when reused. The exact reasons for this observation are currently unknown. Although we did not observe visible biomass after cleaning/sterilization, it is possible that residual biomass remained on the surface at the microscopic level, which could impact the antibiofilm efficacy by interfering with the contact killing mechanism.

Despite excellent inhibitory properties of ABR-MC against bacterial growth and cell clustering, further biofilm accumulation was not completely blocked. As observed in other antimicrobial surfaces, it is possible that contact killing capability of ABR-MC leaves a layer of dead bacteria and other cell components 7, which in turn, can help new bacteria to adhere, while providing a source of nutrients. Nevertheless, biofilm accumulated poorly on ABR-MC 30


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even after 67h. When comparing control surfaces at early time-point (29h or 43h) to the ABR-MC at the later time point (67h), we observed sparse and small bacterial clusters on ABR-MC, suggesting that fewer cells were able to attach and grow thereby delaying the biofilm development process. Furthermore, the EPS-matrix analysis reveals additional structural changes that could explain the defective biofilm accumulation on ABR-MC. For example, microcolonies on control surface at 43h were all surrounded and interconnected by EPS while such structured EPS production was lacking on ABR-MC at 67h, suggesting impaired expression-function of Gtf exoenzymes that are responsible for EPS synthesis by S. mutans. Thus, in addition to antibacterial effects, it is conceivable that ABR-MC may also affect Gtf activity, which would disrupt insoluble EPS glucan production in situ.

A structured EPS-matrix is essential for the biofilm structural stability, cohesiveness, and surface attachment strength 47. Indeed, the underdeveloped biofilm with defective matrix comprised of scattered and amorphous EPS at the interface of ABR-MC (Figures 4 and 5) was readily removed when subjected to low shear forces (0.18 N/m2) (Figure 3). In contrast, biofilms formed on control composite surface harbored a web-like EPS matrix forming a meshwork that tethered large cell clusters into a cohesive and interconnected unit, making them difficult to remove even under the highest shear stress (1.78 N/m2). This feature is relevant in the mouth environment where even exposure to high shear forces (up to 2 N/m2) generated by some oral care instruments are incapable of completely dislodging biofilms 31



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from surfaces due to high viscoelasticity provided by the EPS-matrix 65,66. Thus, weakening the biofilm structural stability by disrupting EPS-matrix assembly could potentially facilitate its removal from ABR-MC surface in the oral cavity.

Having shown promising antibiofilm properties of ABR-MC, further studies are needed to optimize its efficacy and elucidate the molecular mechanisms of action. Specifically, how does the interaction between the test composite and S. mutans cells affect the EPS production in situ. ABR-MC surface chemistry may modulate Gtfs production by S. mutans as well as the binding or enzymatic activity of the secreted exoenzymes on the surface. Simultaneous Gtf activity and gtf gene expression analysis using recently developed time-lapse confocal fluorescent microscopy 67 may elucidate whether and how ABR-MC impact the Gtfs production and function. Furthermore, detailed characterization of ABR-C anchoring, scaffolding and spatial distribution into the resin structure are needed to further understand the non-leachable properties. Since surface wettability, energy and roughness influence both microbial attachment and protein adsorption 20,68,69, characterization of such properties in ABR-MC can provide valuable information to further improve its anti-biofilm effects. Nanoengineering approaches and availability of other promising anti-biofilm materials could lead to development of hybrid composites with enhanced bioactivity. Finally, further evaluation of efficacy and toxicity of ABR-MC using complex mixed-species and in vivo

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rodent caries models are required to determine the potential clinical application of this nonleachable antibiofilm composite.

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Conclusions A new polymerizable, non-leachable imidazolium-containing resin with excellent antibiofilm properties was synthesized via facile and efficient chemistry. Strong antibacterial activity exerted by ABR-C allowed low dose loading (~2%; wt/wt) and tethering into a polymerizable resin formulation (ABR-MC), achieving a good balance between biological and mechanical properties as well as low cytotoxicity. Importantly, ABR-MC impaired biofilm initiation by disrupting bacterial accumulation and cell clustering, and the assembly of EPS matrix, which dampened further biofilm development over time. The resultant biofilm displayed severely reduced biomass and defective architecture lacking a structurally cohesive matrix that was readily detached from the surface. Taken together, the data reveal that ABR-MC may serve as a platform for development of alternative non-leaching antimicrobial materials for dental and other biomedical applications that must contend with biofilms.

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Supporting Information Material Table S1. MIC and MBC of tested compounds.

Table S2. Compositions and cytotoxic property of formulated resins and composites.

Figure S1. (A) 1H-NMR and (B) 13C-NMR spectrums of monoimidazole-containing dimethacrylate resin; (C) 1H-NMR and (D) 13C-NMR spectrums of 3dodecanylimidazoliumbromide dimethacrylate resin (ABR-C).

Figure S2. S. mutans killing activity of control and ABR-MC composites. Images of blood agar plate cultured with S. mutans covered with (A) control or (B) ABR-MC composites. There are no viable S. mutans on the spot covered with ABR-MC composites.

Figure S3. Alternative reaction pathway to polymerizable monoimidazole-monomethacrylate resin.

Figure S4. Binding of Streptococcus mutans to composite surfaces. Representative pseudocolored SEM images of S. mutans bound to (A) control and (B) ABR-MC surfaces, (C) number of bacteria adhered to each surface.

Figure S5. Representative images of skeletonized EPS-matrix. Skeletonized EPS-matrix of (A) control and (B) ABR-MC at each height.

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Figure S6. Schematic diagram of shear-induced biofilm mechanical strength tester (s-BMST). (A) Overview and close-up view of shear-induced biofilm mechanical strength tester, and (B)

schematic diagram of biofilm removal by shear stress.

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Acknowledgement We thank Dr. Anuradha Prokki and Dr. Celine M. Levesque, affiliated with University of Toronto, for the MIC and MBC test on ABR resins. This work was funded in part by Dentsply Sirona.

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Graphical Abstract. Schematic diagram of ABR-C and representative confocal 3D images of 67 h biofilms after exposure to shear stress of 0.80 N/m2. 163x151mm (300 x 300 DPI)


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Figure 1. Schematic diagram of two-pot three-step synthetic route to 3-dodecanylimidazoliumbromide dimethacrylate carbonate (ABR-C). Precursor to ABR-C was prepared via one-pot two-step reaction (Step 1 and 2), and ABR-C was prepared via additional one-pot reaction (Step 3). The circle in Step 3 indicates the incorporated imidazolium moiety into ABR-C. 258x190mm (300 x 300 DPI)



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Figure 2. Dynamics of Streptococcus mutans biofilm formation on each composite surface. (A) Representative confocal images, (B) biovolume (biomass) of EPS and (C) bacteria, (D) total dry-weight, and (E) culture pH of S. mutans biofilms on control and ABR-MC surfaces at various time-points. Bacterial microcolonies labeled with Syto 9 are in green, while EPS labeled with Alexa Fluor 647-dextran are shown in red. Asterisk indicates that the values from ABR-MC are significantly different from control surface (P < 0.05). 175x246mm (300 x 300 DPI)


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Figure 3. Mechanical stability of Streptococcus mutans biofilm on each composite surface. (A) Biofilm removal profile after application of increased shear stress, (B) representative confocal 3D and orthogonal images of 67 h biofilms after exposure to shear stress of 0.81 N/m2. Bacterial microcolonies labeled with Syto 9 are in green, while EPS labeled with Alexa Fluor 647-dextran are shown in red. Asterisk indicates that the biofilm removals from ABR-MC are significantly higher than the values from control surface (P < 0.05). 260x89mm (300 x 300 DPI)



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Figure 4. Representative projection images of EPS-matrix and biofilms on each composite surface. (A1-A3) EPS only and (B1-B3) biofilms (EPS plus bacteria) on control and (C1-C3) EPS and (D1-D3) biofilms on ABRMC surfaces at 29, 43, and 67 h, respectively. Bacterial microcolonies labeled with Syto 9 are in green, while EPS labeled with Alexa Fluor 647-dextran are shown in red. 228x304mm (256 x 256 DPI)


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Figure 5. Representative projection images of skeletonized EPS-matrix from 67h biofilm formed on each composite surface. (A) Overall and (C) close-up views of skeletonized EPS-matrix on control, and (B) overall and (D) close-up views of skeletonized EPS-matrix on ABR-MC surfaces. White arrows indicate the ‘superstructure’ filled with a dense mesh of thin filaments crisscrossing the thicker EPS. 216x205mm (300 x 300 DPI)


Nonleachable Imidazolium-Incorporated ... - ACS Publications

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