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Biological and Medical Applications of Materials and Interfaces

Enhanced Nano-Assembly Incorporated Antimicrobial Composite Materials Lee Schnaider, Moumita Ghosh, Darya Bychenko, Irena Grigoriants, Sarah Ya'ari, Tamar Shalev Antsel, Shlomo Matalon, Rachel Sarig, Tamar Brosh, Raphael Pilo, Ehud Gazit, and Lihi Adler-Abramovich ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02839 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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ACS Applied Materials & Interfaces

Enhanced Nano-Assembly Incorporated Antimicrobial Composite Materials

Lee Schnaider1, Moumita Ghosh2, Darya Bychenko1, Irena Grigoriants2, Sarah Ya’ari2, Tamar Shalev Antsel2, Shlomo Matalon3, Rachel Sarig2, Tamar Brosh2, Raphael Pilo3, Ehud Gazit1,4, Lihi Adler-Abramovich*2

1Department

of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel

Aviv University, Tel Aviv 69978, Israel.2Department of Oral Biology, The Maurice and Gabriela Goldschleger School of Dental Medicine, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.3Department of Oral Rehabilitation, The Maurice and Gabriela Goldschleger School of Dental Medicine, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel.4Department of Materials Science and Engineering, Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel.

*Correspondence to: [email protected]

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ABSTRACT: The rapid advancement of peptide and amino acid based nanotechnology offers new approaches for the development of biomedical materials. The utilization of fluorenylmethyloxycarbonyl (Fmoc)-decorated self-assembling building blocks for antimicrobial and anti-inflammatory purposes represents promising advancements in this field. Here, we present the antibacterial capabilities of the nanoassemblies formed by Fmoc-pentafluoro-L-phenylalanine-OH, their substantial effect on bacterial morphology, as well as new methods developed for the functional incorporation of these nano-assemblies within resin-based composites. These amalgamated materials inhibit and hinder bacterial growth and viability and are not cytotoxic towards mammalian cell lines. Importantly, due to the low dosage required to confer antibacterial activity, the integration of the nano-assemblies does not affect their mechanical and optical properties. This approach expands on the growing number of accounts on the intrinsic antibacterial capabilities of self-assembling building blocks and serves as a basis for further design and development of enhanced composite materials for biomedical applications.

Table of Contents Graphic. Overview of the design scheme of the enhanced nano-assembly incorporated antimicrobial composite restoratives.

KEYWORDS Antimicrobial materials, Self-assembly, Nano-structures, Biomaterials, Resin composite restoratives

INTRODUCTION The self-assembly of peptides and functionalized amino acids into supramolecular nanostructures has yielded a wide variety of nanomaterials with numerous biomedical applications1–5. These include the utilization of the nanostructures as drug delivery platforms6–10 and tissue scaffolds11–17 as well as potent self-assembled antibacterial moieties18–36, including self-assembling antibacterial amphiphilic, cyclic and 2 ACS Paragon Plus Environment

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fibril forming peptides20–28,36. Moreover, self-assembled organic nanomaterials have garnered significant interest due to their chemical diversity, biocompatibility, relatively low synthesis cost, high loading capacity and high purity1–5. Specifically, amino acids and peptides modified with the fluorenylmethyloxycarbonyl (Fmoc) moiety have been demonstrated to form distinct nanostructures whose self-association is strongly governed by the inherent aromaticity and hydrophobicity of the Fmoc moiety37,38. These characteristics promote the hydrophobic and π–π stacking interactions of the fluorenyl rings of the Fmoc group and this building block has been notably utilized to drive hydrogel formation, as well as other nano-morphologies. The resulting assemblies present remarkable physicochemical properties and are utilized for various applications. Particularly, Fmoc-based hydrogels and assemblies are utilized as anti-inflammatory and antibacterial biomaterials26,27,39,40. The influence of side-chain halogenation on the self-assembly and hydrogelation of Fmoc-phenylalanine derivatives has been extensively studied by Nilsson and co-workers who first designed the Fmoc-pentafluoro-phenylalanine (Fmoc-F5-Phe) self-assembling building block, which form hydrogels composed of ordered nanometric fibrils41,42. Research of these hydrogels has primarily focused on their unique physicochemical characteristics, with their mechanical properties greatly improved by the coassembly with the Fmoc-diphenylalanine peptide, resulting in exceptionally stable and rigid materials43. Interestingly, the carbon-fluorine bond of the fluorinated aromatic ring of pentafluoro-phenylalanine has been recently utilized as an antifouling motif35. Furthermore, we have recently evaluated the antibacterial capabilities of the nano-assemblies formed by the diphenylalanine building block, and have found them to have substantial antibacterial and membrane interacting activity24. This work, joined by others demonstrating the antibacterial capabilities of minimal self-assembling building blocks has indeed garnered great interest into the interplay between self-assembly and antimicrobial activity, and provides a new avenue for the development of antibacterial agents and materials24–28,36. The development of such antibacterial compounds and biomedical-relevant materials represents an urgent and unmet medical need44. Specifically, the development of such materials could have a substantial worldwide impact on the field of dental maladies. Dental caries (tooth decay) and periodontal diseases are 3 ACS Paragon Plus Environment


pressing global oral health burdens affecting 60-90% of school-children and the vast majority of adults45– 47.

Dental caries is one of the most prevalent and costly oral diseases caused by the acidification of tooth

enamel and dentin by virulent bacterial species, such as Streptococcus mutans. These bacteria accumulate on the tooth surface and ultimately dissolve the hard tissues of the teeth48. Recurrent caries, also known as secondary tooth decay, at the margins of dental restorations results from acid production by caries-causing bacteria that reside in the restoration-tooth interface. This malady is a major causative factor for dental restorative material failure, and has been estimated to affect over 100 million patients a year, at an estimated cost of over $30 billion45–47. In addition to acid production, enzymes produced by caries-causing bacteria degrade the materials, and the resultant marginal leakage at the restoration-tooth interface contributes to the formation and progression of recurrent caries48. Thus, resin composites containing constituents that also display bacterial inhibitory activity have the potential to substantially hinder the development of these widespread diseases. Several methodologies have been evaluated in the search for such materials, including modification of the resin matrix and/or addition of functional filler particles. The introduction of antibacterial moieties at the restoration site by their incorporation within dental resin-based restorative matrices has been recognized as a key approach in order to prevent bacterial proliferation and secondary caries formation49–54. The incorporated antibacterial moieties can either be released as a soluble agent or remain in the resin in a stationary phase. These moieties are of low molecular weight and of both organic and inorganic nature. The most prominent agents introduced include classical antibiotics, fluoride, chlorhexidine, antibacterial nanomaterials and carriers, silver-based moieties, iodine, zinc and quaternary ammonium compounds49–54. However, the gradual release of soluble agents from the bulk resin has adverse influence on the mechanical properties as the leaching may result in a porous and weak resin49–54. Furthermore, the antibacterial activity in these cases is time-limited and the released compounds may display cytotoxic activity towards the adjacent human tissues49–54. These short comings are amplified when taking into account the relatively high w/w% loading dose needed to effectively inhibit bacterial growth and reduce bacterial viability, which can often reach tens of percentages49–54. 4 ACS Paragon Plus Environment

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Here, in order to address these shortcomings, we demonstrate the antibacterial activity of the Fmocpentafluoro-L-phenylalanine-OH (Fmoc-F5-Phe) self-assembling building block which was chosen as it comprises both functional and structural sub-parts, with the Fmoc-phenylalanine (Fmoc-Phe) group utilized for self-association into ordered assemblies of nanoscale structures, as well as its antibacterial capabilities, and the fluoride decoration utilized for its antibacterial activity and remineralization enhancement capabilities. Furthermore, we present the functional methods developed for incorporating the nanoassemblies formed by Fmoc-F5-Phe within dental resin-based composite restoratives. We present the potent antibacterial capabilities of composite restoratives incorporated with nanostructures formed by Fmoc-F5Phe at an increasing loading dose of up to 2 w/w%, substantially low in comparison to that of other antibacterial dental nano-assemblies, against S. mutans, and demonstrate that the Fmoc-F5-Phe enhanced restoratives are both biocompatible and retain the mechanical strength and optical properties of the original restorative. The identification of the substantial antibacterial capabilities of these minimal self-assembling building blocks provides an important platform for the design and development of additional minimal antimicrobial therapeutics and materials to combat a wide variety of bacterial infections.

RESULTS AND DISCUSSION Generation of a Minimal Antibacterial Building Block. Following solvent-switch based nano-assembly formation of the Fmoc-F5-Phe building block (Figure 1a), flexible, non-branched, fibrillary structures, 25 nm in width were observed via transmission and scanning electron microscopy (Figure 1b-c). Next, we evaluated the antibacterial capabilities of these nano-assemblies against S. mutans via a minimum inhibitory concentration analysis, as well as by kinetic growth inhibition analysis. The nano-assemblies exhibited substantial activity towards S. mutans as overnight incubation at 2mM with cultures that started out at early log-phase at completely inhibited bacterial growth, with a reduction of 7.2 log (10) CFU/ml, with lower concentrations partially inhibiting growth in a dose-dependent manner (Figure 1d). To directly assess bacterial viability, the bacteria were subjected to Live/Dead viability analysis containing Syto9 (indicating 5 ACS Paragon Plus Environment


live bacteria) and propidium iodide (indicating dead bacteria). This analysis revealed that treatment with the nano-assemblies caused significant bacterial cell death (Figure 1e). We next evaluated the ability of the nano-assemblies to inhibit bacterial growth at a higher bacterial load, similar to that of an active infection. S. mutans cultures were grown until mid-log phase and were then treated with 2 mM samples of the nano-assemblies. Kinetic growth inhibition analysis coupled with Live/Dead viability analysis revealed that the nano-assemblies inhibited bacterial proliferation (Figure 1fg). In order to gain insights into the mechanism of action of the nano-assemblies, their effect on bacterial morphology was studied using electron microscopy. Following overnight treatment, membrane fusing, clumping and disintegration were abundant in the treated bacteria, which appeared deflated as compared to the control bacteria (Figure 1h), thus pointing to the bacterial membrane as the main target of these nanoassemblies. The effect of Fmoc-F5-Phe on bacterial membrane permeation was also supported by a SYTOX Blue membrane permeation assay55. SYTOX Blue is a cationic dye which cannot enter into an intact cell unless its membrane is disrupted by external compounds. Inside the cell, SYTOX Blue stain binds to intracellular nucleic acids and fluoresce bright blue when excited with 405 nm violet laser light. We found significant enhancement in the fluorescence of bacterial samples treated with Fmoc-F5-Phe - about 90% as opposed to the control sample, in which less than 1% were stained with this dye (Supplementary Figure 1). Taken together, these results demonstrate the substantial membrane disruption capabilities of the nanoassemblies.


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Figure 1. Characterization of the Potent Antibacterial Capabilities of the Nano-Assemblies Formed by Fmoc-F5-Phe. (a) Molecular structure of the Fmoc-F5-Phe building block. (b-c) Nano-assemblies formed by Fmoc-F5-Phe. Micrographs were obtained using (b) transmission electron microscopy, scale bar is 2μm and (c) scanning electron microscopy, scale bar is 1μm. (d) Bacterial growth inhibition kinetics evaluated by turbidity analysis via absorbance readings at 650 nm. (e) Bacterial viability evaluation following overnight growth inhibition kinetic analysis using the Live/Dead backlight bacterial viability kit. Green fluorescence of the Syto9 probe indicates bacterial cells with an intact membrane, while the red fluorescence of propidium iodide (PI) indicates dead bacterial cells. (f-g) Effect of treatment with the nano-assemblies on mid-log phase bacteria. (f) Bacterial growth inhibition kinetics following the addition of the nano-assemblies to mid-log phase bacteria, evaluated by turbidity analysis via absorbance readings at 650 nm. (g) Bacterial viability evaluation following four-hour incubation of mid-log phase bacteria with the nano-assemblies. Green fluorescence of the Syto9 probe indicates bacterial cells with an intact membrane, while the red fluorescence of propidium iodide (PI) indicates dead bacterial cells. (h) Evaluation of the effect of the nano-assemblies on bacterial morphology. Micrographs were obtained using a high-resolution scanning electron microscope. Scale bar is 1 µm.



Development of Enhanced Composite Antibacterial Restoratives. Once the antibacterial activity of the Fmoc-F5-Phe nano-assemblies was established, we explored the ability of these assemblies to confer their antibacterial capabilities to dental resin composite restoratives while maintaining the biocompatibility, mechanical stability and optical properties of the original restorative. Following assembly formation, the nano-assemblies were incorporated into the pre-polymerized Filtek™ Ultimate Flow dental resin composite restorative (3M-ESPE), a widely used restorative material which does not display inherent antimicrobial capabilities56, by manual mixing, sonication and centrifugation. The amalgamated resin composite was subsequently polymerized by visible blue light. This incorporation process yielded a uniform and even distribution of the nano-assemblies within the amalgamated restorative, as demonstrated by energydispersive X-ray spectroscopy (EDX) analysis and optical microscopy (Supplementary Figure 2). The particular size of the nano-assemblies formed by this building block allows for their facile and functional incorporation into the dental resin composites used as clinical restorative materials. In order to evaluate the antimicrobial capabilities of the resin composite restoratives while simulating its clinical use, a direct-contact test57 (DCT) was carried out. This spectroscopic microplate-reader based test, designed for compounds that are non-diffusible and non-soluble in water, allows to measure the effect of direct contact between the evaluated material and bacterial viability and growth. Four different samples of the resin composite material incorporated with either 0.25, 0.5, 1 or 2 w/w% of the nano-assemblies were evaluated, along with Filtek™ Ultimate Flow with no Fmoc-F5-Phe nano-assembly additives, treated in the same manner, which served as a control. Following direct contact of S. mutans bacteria with the nanoassembly incorporated materials, bacterial proliferation was evaluated by optical density measurements over eighteen hours. The samples containing 0.25-1% nano-assemblies were able to partially inhibit bacterial growth in a dose dependent manner, while the 2% nano-assembly samples were able to cause substantial (over 95%) bacterial growth inhibition (Figure 2a) and bacterial cell death, as evidenced by Live/Dead bacterial viability analysis (Figure 2b).


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Biocompatibility of the Enhanced Composite Antibacterial Restoratives. In order to examine the biocompatibility of the resin composite restorative incorporated with the Fmoc-F5-Phe nano-assemblies, an MTT-based cell viability assay was carried out. 3T3 fibroblasts and HeLa cells were grown overnight in 96 well plates in the presence of the nano-assembly incorporated resin composite restorative, as well as the Filtek™ Ultimate Flow material treated in the same manner as a control. Cell viability was not significantly altered by the presence of the nano-assembly incorporated resin composite restorative, as compared to the control (Figure 2c-d). Similar results were obtained using Live/Dead staining, containing fluorescein diacetate (indicating live cells) and Propidium Iodide (indicating dead cells) of cells treated in the same manner (Figure 2e-f). These results indicate the enhanced antibacterial potency of the amalgamated resin composite restorative, as the cytotoxic activity is not directed towards mammalian cell lines but only towards bacterial cells. While several restorative and resin-based materials have been embedded with bioactive compounds, a high-dose loading of these compounds is usually required to achieve their antibacterial activity, resulting in low biocompatibility. The low dosage needed to achieve successful antibacterial activity of the Fmoc-F5-Phe nano-assemblies a well as their inherent tendency to self-assemble, a trait well documented to reduce cytotoxicity towards mammalian cells, are key factors in the biocompatibility of these resin composite restoratives.

Mechanical and Optical Properties of the Enhanced Composite Antibacterial Restoratives. Two important factors, which may preclude clinical use of the nano-assembly incorporated resin composite restorative, are their mechanical stability and optical properties. In order to assure that the integration of the Fmoc-F5-Phe nano-assemblies does not weaken the composite mechanical properties, a shear punch strength (SPS) test and diametral tensile test (DTS) were conducted.



Figure 2. Antibacterial Capabilities and Biocompatibility of the Enhanced Resin Composite Restoratives. (a) Bacterial growth inhibition kinetics evaluated by turbidity analysis via absorbance readings at 650 nm following direct contact of S. mutans bacteria with the Fmoc-F5-Phe incorporated restoratives for one hour. (b) Bacterial viability evaluation following direct contact analysis with either the Filtek™ resin composite restorative or Fmoc-F5-Phe nanoassembly incorporated resin composite restorative using the Live/Dead backlight bacterial viability kit. (c-d) MTT cell viability analysis. The cytotoxicity of the Fmoc-F5-Phe incorporated restoratives toward (c) 3T3 fibroblast and (d) HeLa cells was evaluated by the MTT assay following overnight incubation with the nano-assembly incorporated restoratives at 2% w/w% as well as control restoratives treated in the same manner that were not incorporated with nano-assemblies (e-f) Mammalian cell viability analysis following overnight incubation with the nano-assemblies incorporated and control restoratives was carried out utilizing a fluorescent Live/Dead staining assay containing fluorescein diacetate (staining live cells) and propidium iodide (indicating dead cells). (e) 3T3 fibroblasts; (f) HeLa cells. Scale bar is 500 µm.

The punch shear test was utilized as it has been shown that during mastication, occlusal forces induce shear stresses within both teeth and restorations58,59. The reliability of the test is reflected by a low coefficient of variation of approximately 8%. No statistically significant difference was found between the samples containing 0.25, 0.5 or1 w/w% of the nano-assemblies as compared to the control (0%) (p≥0.144), while


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the sample containing 2% of the nano-assemblies was found to slightly differ from the control (p=0.011), though to an extent of no clinical significance (Figure 3a). The DTS test was further performed to verify the difference in tensile properties (tensile strength and stiffness of the specimens characterizing the elasticity of the materials) of the 2% amalgamated material as compared to the control (0%) (Figure 3b). No statistically significant differences were found in either strength or stiffness (p>0.155). The inherent stability of the amalgamated materials was next demonstrated via an HPLC-based nano-assembly leeching evaluation which was carried out over 72 hours in sterile salvia (Supplementary Table 1). Additionally, large occlusal fissures restorations performed with both the 2% nano-assembly amalgamated restoratives and the control (0%) remained intact and stable following a 30-day incubation at 37°C in sterile PBS (Figure 3c). The effect of nano-assembly incorporation on the optical properties of the amalgamated restoratives, an esthetically important feature for their clinical use, was evaluated utilizing a Spectroshade Micro – MHT dental spectrophotometer normalized to the Vita classical color guide. Both the 2% nano-assembly amalgamated restoratives and the control (0%) were spectroscopically identified to be of the same Vita shade (Figure 3d). The results of the optical and mechanical evaluation can be attributed to the size of the Fmoc-F5-Phe nano-assemblies and the low loading dose required for their conferral of antimicrobial activity to the resin composite restoratives.



Figure 3. Mechanical and Optical Properties of the Enhanced Resin Composite Restoratives. (a-b) Evaluation of the mechanical stability of the Fmoc-F5-Phe incorporated restoratives by (a) shear punch strength analysis and (b) diametral tensile strength analysis. Fmax represents the maximum applied force required to physically punch through each sample. (c-d) Evaluation of the effect of the incorporation of the nano-assemblies on the optical properties of the enhanced resin composite restoratives. (c) Restoration of occlusal fissures with the control (left) and nano-assembly incorporated restoratives (right). (d) Spectral characterization of the color of the control (left) and nano-assembly incorporated restoratives (right) utilizing a Spectroshade Micro – MHT dental spectrophotometer normalized to the Vita classical color guide.

CONCLUSIONS In conclusion, we have demonstrated the antibacterial activity of the nano-assemblies formed by the FmocF5-Phe building block and the functional incorporation of these nano-assemblies into dental resin composite restorative, thus successfully generating enhanced antibacterial and biocompatible resin composite amalgamated materials. These enhanced composites require only 2% w/w% loading of the antibacterial nano-assemblies, thus allowing the amalgamated material to maintain the mechanical strength and optical properties of the original composite. These results provide important insights into the intrinsic antibacterial capabilities of minimal self-assembling building blocks commonly utilized for structural and physical applications, a phenomenon that has only recently been identified and holds great potential for numerous nanoscience and biomedical applications. The minimal nature of the Fmoc-F5-Phe antibacterial building


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block, along with its high purity, low cost, ease of embedment within resin-based materials and biocompatibility, allows for the facile scale-up of this approach towards the development of clinically available enhanced antibacterial resin composite restoratives.

METHODS Nano-assembly formation. The lyophilized powder of Fmoc-pentafluoro-phenylalanine (Fmoc-F5-Phe) was purchase from Chem-Impex International Inc. Nano-assemblies were prepared by the solvent switch method. First, to generate a stock solution, the powder was dissolved to 10 mg/ml in ethanol, followed by x10 dilution with double distilled water to a final concentration of 1 mg/ml. Immediately after dilution, the resulting solution was strongly mixed by vortex and incubated overnight under ambient conditions to allow self-assembly processes. The formed nano-assemblies were lyophilized overnight with the resulting ethanol concentration in these samples effectively zero due to the lyophilization process. High-resolution scanning electron microscopy of nano-assemblies. Following nano-assembly formation, 10 µg of the lyophilized powder was deposited on carbon conductive adhesive tape and samples were then coated with Chromium (Cr). Micrographs were recorded using a JSM-6700F FE-SEM (JEOL, Tokyo, Japan) operating at 5 kV. Fabrication of nano-assembly incorporated resin composite restoratives. The Fmoc-F5-Phe nanoassemblies were incorporated into the Filtek™ Ultimate Flow dental resin composite restorative (3M, ESPE), while ensuring that the structures are efficiently and evenly distributed. Nano-assemblies were added to the commercial pre-polymerized material at four different concentrations: 0.25, 0.5, 1 and 2 w/w%. Each sample was centrifuged for 1 minute at 3700 RPM and then mixed manually for three minutes followed by 1-minute centrifugation at 3700 RPM, and sonication for 5 minutes. Following sonication, samples were centrifuged for 1 minute, manually mixed for three minutes and then centrifuged for 1 minute



at 3700 RPM. The resulting amalgamated restoratives were polymerized for 40 seconds per individual sample, by Elipar Trilight (3M, ESPE), a high-performance light polymerization unit for dental materials which are polymerized under visible blue light in the mouth. Energy-dispersive X-ray spectroscopy (EDX). Nano-assembly incorporated resin composite restoratives were light cured on glass slides and imaged using a JSM-6700F FEG-SEM (JEOL, Tokyo, Japan). EDX analysis using Oxford INCA (Oxford Instrument America Inc., Concord, MA, USA) was carried out on the visualized sample area. Kinetic growth inhibition and MIC analysis. S. mutans bacteria were grown under anaerobic conditions in BHI medium (BD Difco) for 48 hours and then diluted to OD600 of 0.01 or 0.25 in BHI. Nano-assembly samples, at an initial concentration of 8mM, were added to the bacterial samples in 96-well plates in serial two-fold dilutions, which were sealed to ensure anaerobic conditions. Kinetic growth inhibition was determined by optical density measurements (650 nm) using a Biotek Synergy HT microplate reader. The MIC was determined using the microdilution assay and evaluation of the reduction in colony forming units was obtained by plating and counting bacterial samples before and after overnight treatment. The MIC was considered the lowest peptide concentration that showed no increase in optical density and no CFU growth overnight. Presented kinetic analysis and MIC results are representative of three independent experiments conducted in quadruplets. Bacterial viability analysis. Following kinetic analysis, samples were washed thrice with saline, incubated for 15 min in a solution containing Syto9 and propidium iodide (L13152 LIVE/DEAD® BacLight™ Bacterial Viability Kit, Molecular Probes, OR, USA) and washed with saline again. Fluorescence emission was detected using an ECLIPSE E600 fluorescent microscope (Nikon, Japan). The presented results are representative of three independent experiments.


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High-resolution scanning electron microscopy of bacterial samples. Bacterial samples were centrifuged at 5000 RPM for 5 min, washed thrice in PBS and fixed in 2.5% glutaraldehyde in PBS for 1 h. Samples were then washed thrice in PBS and fixed in 1% OsO4 in PBS for 1 h, followed by a dehydration series with ethanol. Samples were then left in absolute ethanol for 30 min and placed onto glass coverslips, followed by critical point drying and coating with gold. Micrographs were recorded using a JEOL JSM6700F FE-SEM scanning electron microscope operating at 10 kV. Presented micrographs are representative of three independent experiments. Membrane permeation assay. S. mutans bacteria were grown under anaerobic conditions in BHI medium (BD Difco) for 48 and diluted to 0.1 O.D.600. 100 μl of Fmoc-F5-Phe or ultra-pure water as control were added to 300 μl of bacteria and incubated for 3 hours in 37 °C. The bacteria cells were centrifuged for 5 minutes at 3700 rpm and were incubated with 1 μM SYTOX blue (ThermoFisher Scientific) for 30 minutes in 37 °C. Samples were washed three times in PBS and examined by confocal microscopy LSM 510, excited at 405 nm (Zeiss, Germany). Direct-contact kinetic analysis. S. mutans bacteria were grown under anaerobic conditions in BHI medium (BD Difco) for 48 hours and then diluted to OD600 0.6 in BHI. A modified Direct Contact Test was carried out as follows: 10µl of each sample were deposited onto inserts (concaved plastic surfaces designed to be suspended in the wells of 96 well-plates) coated on one side with the nano-assembly incorporated resin composite restoratives and then incubated for one hour at 37 C. Following incubation, 225µl of BHI was added to each well so that the inserts were submerged in the media and the plates were sealed to ensure anaerobic conditions. Kinetic growth inhibition was determined by optical density measurements (650 nm) using a Biotek Synergy HT microplate reader. Presented kinetic analysis results are representative of three independent experiments conducted in quadruplets. Shear punch strength test. A shear punch test was performed according to Mount and coworkers60–62. Briefly, pre-polymerized nano-assembly incorporated resin composite restoratives were placed in 0.8mm 15 ACS Paragon Plus Environment


thick wash holders and light-cured to form flat parallel surfaces evenly supported and restrained by the holder (as demonstrated to be an important prerequisite by Pilo and coworkers63). Samples were then placed in an Instron device (model 4502) for punching under cross-head speed of 0.5mm per minute. The maximum force applied (Fmax) was calculated as the mean of ten different samples for each w/w% concentration of Fmoc-F5-Phe. Statistical analysis was carried out via one-way analysis of variance anda Dunnett Post Hoc Test. Diametral tensile strength (DTS) test: Disks (6 mm in diameters, 3 mm in height) of either control resin composite restorative or 2% nano-assembly incorporated resin composite restorative were prepared in a Teflon mold similar to the specimens used for the punch shear strength. Specimens were loaded up to failure. The linear slope during loading was calculated, indicating the stiffness of the specimen and the DTS 2𝑃

was calculated by: DTS=2P/𝐷𝑇𝑆 = 𝜋Dt where P is the load at failure (N), D – specimen diameter (mm), and t – specimen height (mm). The specimens were loaded via the above mentioned loading machine using the same crosshead speed. Statistical analysis was carried out via T-test. Mammalian cell cytotoxicity experiments. 3T3 mouse fibroblasts and HeLa cells grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel) were sub-cultured (2 × 105 cells/mL) in 96-well tissue microplates (100 µl per well) and allowed to adhere overnight at 37°Cin a humidified atmosphere containing 5% CO2. Quadruplet inserts coated on one side with the nano-assembly incorporated resin composite restoratives were placed into the wells containing the adhered cells. After incubation for 18 hours at 37°C, cell viability was evaluated using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 10 µL of 5 mg/mL MTT dissolved in PBS was added to each well. After a 4-hour incubation at 37°C, 100 µl extraction buffer [20% SDS dissolved in a solution of 50% dimethylformamide and 50% DDW (pH 4.7)] was added to each well, and the plates were incubated again at 37°C for 30 minutes. Finally, color intensity was measured using an ELISA reader at 570 nm. The presented results are the mean of three independent experiments.


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Mammalian cell viability analysis. 3T3 mouse fibroblasts and Hela cells were cultured in DMEM supplemented with 10% FBS, 100 U mL-1 penicillin, 100 U mL-1 streptomycin, and 2 mmol L-1 L-glutamine (all from Biological Industries, Israel). The cells were cultured in a petri dish at 37 °C in a humidified atmosphere containing 5% CO2. After reaching 90%confluence, the cells were dissociated from the petri dish using trypsin A and 10,000 cells in 0.1 ml of fresh culture medium were seeded per well of a 96 wellplate and kept overnight for attachment at 37 °C in a humidified atmosphere containing 5% CO2. Quadruplet inserts coated on one side with the nano-assembly incorporated resin composite restoratives were placed into the wells containing the adhered cells, followed by a15-hour incubation. A fluorescent Live/Dead staining assay (Sigma Aldrich) containing fluorescein diacetate (6.6 µg/ml) and propidium iodide (5 µg/ml) was then used to visualize the proportion of viable versus non-viable cells in each sample. The labelled cells were immediately viewed using a Nikon Eclipse Ti fluorescent microscope and images were captured by a Zyla scMOS camera using Nikon Intensilight C-HGFI fluorescent lamp. The presented results are representative of three independent experiments. Occlusal Fissure Stability and Optical Property Analyses. Occlusal fissures were made via a diamond bur and then restored utilizing either the 2% nano-assembly amalgamated restoratives or the control (0%) restorative. Samples were contained for 30 days at 37°C in sterile PBS. A Spectroshade Micro – MHT dental spectrophotometer normalized to the Vita classical color guide was then utilized to evaluate the color of the control and amalgamated restoratives.

SUPPLEMENTARY INFORMATION The supplementary information is available free of charge. Membrane permeation analysis, Homogenous incorporation of the Fmoc-F5-Phe nano-assemblies within the resin composite restorative, Stability study of the antibacterial resin composite restorative.



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ACKNOWLEDGMENTS This work is dedicated to the memory of the remarkable Dr. Anszel Sznajder, DMD. We thank Ariel Pokhojaev for his assistance with the analysis of the optical properties of the amalgamated restoratives; Zohar A. Arnon for his assistance in figure preparation; Noam Brown for his assistance in EDX analysis; Dr. Vered Holdengreber for her assistance in preparation of bacterial samples for electron microscopy and Dr. Sigal Rencus-Lazar for her assistance in manuscript editing. We thank the members of the Adler-Abramovich and Gazit groups for fruitful discussions. This project was partially supported by a grant from the ITI Foundation, Switzerland (L.A-A), and a scholarship administered by the Israeli 23 ACS Paragon Plus Environment


Ministry of Science, Technology and Space (L.S.). The authors acknowledge the Chaoul Center for Nanoscale Systems of Tel Aviv University for the use of instruments and staff assistance.

COMPETING FINANCIAL INTERESTS The authors declare no conflict of interest.


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