Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 21334−21342
www.acsami.org
Enhanced Nanoassembly-Incorporated Antibacterial 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*,‡
Downloaded via UNIV OF SOUTHERN INDIANA on July 17, 2019 at 08:01:37 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, ‡Department of Oral Biology, The Maurice and Gabriela Goldschleger School of Dental Medicine, Sackler Faculty of Medicine, §Department of Oral Rehabilitation, The Maurice and Gabriela Goldschleger School of Dental Medicine, Sackler Faculty of Medicine, and ∥Department of Materials Science and Engineering, Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel S Supporting Information *
ABSTRACT: The rapid advancement of peptide- and aminoacid-based nanotechnology offers new approaches for the development of biomedical materials. The utilization of fluorenylmethyloxycarbonyl (Fmoc)-decorated self-assembling building blocks for antibacterial 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 nanoassemblies within resin-based composites. These amalgamated materials inhibit and hinder bacterial growth and viability and are not cytotoxic toward mammalian cell lines. Importantly, due to the low dosage required to confer antibacterial activity, the integration of the nanoassemblies 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. KEYWORDS: antibacterial materials, self-assembly, nanostructures, biomaterials, resin composite restoratives
■
biomaterials.26,27,39,40 The influence of side-chain halogenation on the self-assembly and hydrogelation of Fmoc-phenylalanine derivatives has been extensively studied by Nilsson and coworkers, who first designed the Fmoc-pentafluoro-phenylalanine (Fmoc-F5-Phe) self-assembling building block, which forms hydrogels composed of ordered nanometric fibrils.41,42 Research on these hydrogels has primarily focused on their unique physicochemical characteristics, with their mechanical properties greatly improved by the co-assembly with the Fmocdiphenylalanine peptide, resulting in exceptionally stable and rigid materials.43 Interestingly, the carbon−fluorine bond of the fluorinated aromatic ring of pentafluoro-phenylalanine has been recently utilized as an antifouling motif.35 Furthermore, we have recently evaluated the antibacterial capabilities of the nanoassemblies formed by the diphenylalanine building block and have found them to have substantial antibacterial and membrane interacting activity.24 This work, joined by others demonstrating the antibacterial capabilities of minimal selfassembling building blocks has indeed garnered great interest into the interplay between self-assembly and antibacterial
INTRODUCTION The self-assembly of peptides and functionalized amino acids into supramolecular nanostructures has yielded a wide variety of nanomaterials with numerous biomedical applications.1−5 These include the utilization of the nanostructures as drugdelivery platforms6−10 and tissue scaffolds11−17 as well as potent self-assembled antibacterial moieties,18−36 including self-assembling antibacterial amphiphilic, cyclic, and fibrilforming peptides.20−28,36 Moreover, self-assembled organic nanomaterials have garnered significant interest due to their chemical diversity, biocompatibility, relatively low synthesis cost, high loading capacity, and high purity.1−5 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 moiety.37,38 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 nanomorphologies. 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 © 2019 American Chemical Society
Received: February 14, 2019 Accepted: May 28, 2019 Published: May 28, 2019 21334
DOI: 10.1021/acsami.9b02839 ACS Appl. Mater. Interfaces 2019, 11, 21334−21342
Research Article
ACS Applied Materials & Interfaces
Figure 1. Characterization of the potent antibacterial capabilities of the nanoassemblies formed by Fmoc-F5-Phe. (a) Molecular structure of the Fmoc-F5-Phe building block. (b, c) Nanoassemblies formed by Fmoc-F5-Phe. Micrographs were obtained using (b) transmission electron microscopy (scale bar = 2 μm) and (c) scanning electron microscopy (scale bar = 1 μm). (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 nanoassemblies on mid-log-phase bacteria. (f) Bacterial growth inhibition kinetics following the addition of the nanoassemblies to mid-log-phase bacteria, evaluated by turbidity analysis via absorbance readings at 650 nm. (g) Bacterial viability evaluation following 4 h incubation of mid-log-phase bacteria with the nanoassemblies. 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 nanoassemblies on bacterial morphology. Micrographs were obtained using a high-resolution scanning electron microscope. The scale bar is 1 μm.
medical need.44 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 pressing global oral health burdens affecting 60−
activity, and provides a new avenue for the development of antibacterial agents and materials.24−28,36 The development of such antibacterial compounds and biomedical-relevant materials represents an urgent and unmet 21335
DOI: 10.1021/acsami.9b02839 ACS Appl. Mater. Interfaces 2019, 11, 21334−21342
Research Article
ACS Applied Materials & Interfaces 90% of school children and the vast majority of adults.45−47 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 teeth.48 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 billion.45−47 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 caries.48 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 to prevent bacterial proliferation and secondary caries formation.49−54 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 compounds.49−54 However, the gradual release of soluble agents from the bulk resin has an adverse influence on the mechanical properties as the leaching may result in a porous and weak resin.49−54 Furthermore, the antibacterial activity in these cases is timelimited and the released compounds may display cytotoxic activity toward the adjacent human tissues.49−54 These shortcomings 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 percentages.49−54 Here, to overcome these shortcomings, we demonstrate the antibacterial activity of the Fmoc-pentafluoro-L-phenylalanineOH (Fmoc-F5-Phe) self-assembling building block, which was chosen as it comprises both functional and structural subparts, 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-F5-Phe at an increasing loading dose of up to 2 w/w %, substantially low in comparison to that of other antibacterial dental nanoassemblies, 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 antibacterial 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 nanoassembly formation of the Fmoc-F5-Phe building block (Figure 1a), flexible, nonbranched, fibrillary structures of 25 nm width were observed via transmission and scanning electron microscopy (Figure 1b,c). Next, we evaluated the antibacterial capabilities of these nanoassemblies against S. mutans via a minimum inhibitory concentration (MIC) analysis, as well as by kinetic growth inhibition analysis. These nanoassemblies exhibited substantial activity toward S. mutans as overnight incubation at 2 mM with cultures that started out at early log phase 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 live bacteria) and propidium iodide (PI) (indicating dead bacteria). This analysis revealed that treatment with the nanoassemblies caused significant bacterial cell death (Figure 1e). We next evaluated the ability of the nanoassemblies 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 nanoassemblies. Kinetic growth inhibition analysis coupled with Live/Dead viability analysis revealed that the nanoinhibited bacterial proliferation at these concentrations (Figure 1f,g). To gain insights into the mechanism of action of the nanoassemblies, 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 compared to the control bacteria (Figure 1h), thus pointing to the bacterial membrane as a target of these nanoassemblies. The effect of Fmoc-F5-Phe on bacterial membrane permeation was also supported by a SYTOX Blue membrane permeation assay.55 SYTOX Blue is a cationic dye that cannot enter an intact cell unless its membrane is disrupted by external compounds. Inside the cell, SYTOX Blue stain binds to intracellular nucleic acids and fluoresces bright blue when excited with 405 nm violet laser light. We found significant enhancement in the fluorescence of bacterial samples treated with Fmoc-F5-Pheabout 90%as opposed to the control sample, in which less than 1% were stained with this dye (Supporting Information Figure S1). Taken together, these results demonstrate the substantial membrane disruption capabilities of the nanoassemblies. Development of Enhanced Composite Antibacterial Restoratives. Once the antibacterial activity of the Fmoc-F5Phe nanoassemblies 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 nanoassemblies were incorporated into the prepolymerized 21336
DOI: 10.1021/acsami.9b02839 ACS Appl. Mater. Interfaces 2019, 11, 21334−21342
Research Article
ACS Applied Materials & Interfaces
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 1 h. (b) Bacterial viability evaluation following direct contact analysis with either the Filtek resin composite restorative or Fmoc-F5Phe nanoassembly-incorporated resin composite restorative using the Live/Dead backlight bacterial viability kit. (c, d) 3-(4,5-Dimethylthiazolyl-2)2,5-diphenyltetrazolium bromide (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 nanoassembly-incorporated restoratives at 2 w/w % as well as control restoratives treated in the same manner that were not incorporated with nanoassemblies (e, f) Mammalian cell viability analysis following overnight incubation with the nanoassemblies incorporated and control restoratives was carried out utilizing a fluorescent Live/ Dead staining assay containing fluorescein diacetate (indicating live cells) and propidium iodide (indicating dead cells). (e) 3T3 fibroblasts and (f) HeLa cells. The scale bar is 500 μm.
of direct contact between the evaluated material and bacterial viability and growth. Four different samples of the resin composite material incorporated with 0.25, 0.5, 1, or 2 w/w % of the nanoassemblies were evaluated, along with Filtek Ultimate Flow with no Fmoc-F5-Phe nanoassembly 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 18 h. The samples containing 0.25−1% nanoassemblies were able to partially inhibit bacterial growth in a dose-dependent manner, while the 2% nanoassembly 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). Biocompatibility of the Enhanced Composite Antibacterial Restoratives. To examine the biocompatibility of the resin composite restorative incorporated with the Fmoc-F5-
Filtek Ultimate Flow dental resin composite restorative (3MESPE), a widely used restorative material that does not display inherent antibacterial capabilities,56 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 nanoassemblies within the amalgamated restorative, as demonstrated by energy-dispersive X-ray spectroscopy (EDX) analysis and optical microscopy (Supporting Information Figure S2). The particular size of the nanoassemblies formed by this building block allows for their facile and functional incorporation into the dental resin composites used as clinical restorative materials. To evaluate the antibacterial capabilities of the resin composite restoratives while simulating its clinical use, a direct contact test57 was carried out. This spectroscopic microplatereader-based test, designed for compounds that are nondiffusible and nonsoluble in water, allows to measure the effect 21337
DOI: 10.1021/acsami.9b02839 ACS Appl. Mater. Interfaces 2019, 11, 21334−21342
Research Article
ACS Applied Materials & Interfaces
Figure 3. Mechanical and optical properties of the enhanced resin composite restoratives. (a, b) Evaluation of the mechanical stability of the FmocF5-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 nanoassemblies on the optical properties of the enhanced resin composite restoratives. (c) Restoration of occlusal fissures with the control (left) and nanoassemblyincorporated restoratives (right). (d) Spectral characterization of the color of the control (left) and nanoassembly-incorporated restoratives (right) utilizing a Spectroshade Micro-MHT dental spectrophotometer normalized to the Vita classical color guide.
samples containing 0.25, 0.5, or 1 w/w % of the nanoassemblies compared to the control (0%) (p ≥ 0.144), while the sample containing 2% of the nanoassemblies 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 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 a high performance liquid chromatography-based nanoassembly leeching evaluation, which was carried out over 72 h in sterile salvia (Supporting Information Table S1). Additionally, large occlusal fissure restorations performed with both the 2% nanoassemblyamalgamated restoratives and the control (0%) remained intact and stable following a 30 day incubation at 37 °C in sterile phosphate buffered saline (PBS) (Figure 3c). The effect of nanoassembly 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% nanoassembly-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 nanoassemblies and the low loading dose required for their conferral of antibacterial activity to the resin composite restoratives.
Phe nanoassemblies, 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 nanoassembly-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 nanoassembly-incorporated resin composite restorative, 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 toward mammalian cell lines but only toward 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 nanoassemblies as well as their inherent tendency to self-assemble, a trait well documented to reduce cytotoxicity toward 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 nanoassemblyincorporated resin composite restorative, are their mechanical stability and optical properties. To assure that the integration of the Fmoc-F5-Phe nanoassemblies does not weaken the composite mechanical properties, a shear punch strength test and diametral tensile strength (DTS) were conducted. The punch shear test was utilized as it has been shown that during mastication, occlusal forces induce shear stresses within both teeth and restorations.58−62 The reliability of the test is reflected by a low coefficient of variation of approximately 8%. No statistically significant difference was found between the
■
CONCLUSIONS In conclusion, we have demonstrated the antibacterial activity of the nanoassemblies formed by the Fmoc-F5-Phe building block and the functional incorporation of these nanoassemblies into dental resin composite restoratives, thus successfully generating enhanced antibacterial and biocompatible resin 21338
DOI: 10.1021/acsami.9b02839 ACS Appl. Mater. Interfaces 2019, 11, 21334−21342
Research Article
ACS Applied Materials & Interfaces
increase in optical density and no colony forming unit (CFU) growth overnight. Presented kinetic analysis and MIC results are representative of three independent experiments conducted in quadruplets. Bacterial Viability Analysis. Following kinetic analysis, the 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), 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. 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. The samples were then washed thrice in PBS and fixed in 1% OsO4 in PBS for 1 h, followed by a dehydration series with ethanol. The 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 JSM-6700F FE-SEM scanning electron microscope operating at 10 kV. The 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 h and diluted to 0.1 OD600. Fmoc-F5-Phe or ultrapure water (100 μL) as control was added to 300 μL of bacteria and incubated for 3 h in 37 °C. The bacterial cells were centrifuged for 5 min at 3700 rpm and incubated with 1 μM SYTOX blue (Thermo Fisher Scientific) for 30 min at 37 °C. The 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 h and then diluted to OD600 0.6 in BHI. A modified direct contact test was carried out as follows: 10 μL of each sample was deposited onto inserts (concave plastic surfaces designed to be suspended in the wells of 96-well plates) coated on one side with the nanoassemblyincorporated resin composite restoratives and then incubated for 1 h at 37 °C. Following incubation, 225 μL 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. The 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 co-workers.60−62 Briefly, prepolymerized nanoassembly-incorporated resin composite restoratives were placed in 0.8 mm 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). The samples were then placed in an Instron device (model 4502) for punching under a crosshead speed of 0.5 mm per min. The maximum force applied (Fmax) was calculated as the mean of 10 different samples for each w/w % concentration of Fmoc-F5-Phe. Statistical analysis was carried out via one-way analysis of variance and Dunnett’s post hoc test. Diametral Tensile Strength (DTS) Test. Disks (6 mm in diameter, 3 mm in height) of either control resin composite restorative or 2% nanoassembly-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 was calculated by: 2P DTS = 2P /DTS = πDt , where P is the load at failure (N), D is the specimen diameter (mm), and t is the 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.
composite-amalgamated materials. These enhanced composites require only 2 w/w % loading of the antibacterial nanoassemblies, 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 selfassembling 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 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 toward the development of clinically available enhanced antibacterial resin composite restoratives.
■
METHODS
Nanoassembly Formation. The lyophilized powder of Fmocpentafluoro-phenylalanine (Fmoc-F5-Phe) was purchased from Chem-Impex International Inc. Nanoassemblies 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 10× 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 nanoassemblies were lyophilized overnight with the resulting ethanol concentration in these samples effectively zero due to the lyophilization process. High-Resolution Scanning Electron Microscopy of Nanoassemblies. Following nanoassembly 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 Nanoassembly-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. Nanoassemblies were added to the commercial prepolymerized material at four different concentrations: 0.25, 0.5, 1, and 2 w/w %. Each sample was centrifuged for 1 min at 3700 rpm and then mixed manually for 3 min, followed by 1 min centrifugation at 3700 rpm and sonication for 5 min. Following sonication, the samples were centrifuged for 1 min, manually mixed for 3 min, and then centrifuged for 1 min at 3700 rpm. The resulting amalgamated restoratives were polymerized for 40 s 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). Nanoassemblyincorporated 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 Instruments America, Inc., Concord, MA) was carried out on the visualized sample area. Kinetic Growth Inhibition and MIC Analysis. S. mutans bacteria were grown under anaerobic conditions in brain heart infusion (BHI) medium (BD Difco) for 48 h and then diluted to OD600 of 0.01 or 0.25 in BHI. Nanoassembly samples, at an initial concentration of 8 mM, were added to the bacterial samples in 96well plates in serial 2-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 minimum inhibitory concentration (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 21339
DOI: 10.1021/acsami.9b02839 ACS Appl. Mater. Interfaces 2019, 11, 21334−21342
ACS Applied Materials & Interfaces 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 subcultured (2 × 105 cells/mL) in 96-well tissue microplates (100 μL per well) and allowed to adhere overnight at 37 °C in a humidified atmosphere containing 5% CO2. Quadruplet inserts coated on one side with the nanoassemblyincorporated resin composite restoratives were placed into the wells containing the adhered cells. After incubation for 18 h at 37 °C, cell viability was evaluated using the 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (MTT) assay. Briefly, 10 μL of 5 mg/ mL MTT dissolved in PBS was added to each well. After a 4 h incubation at 37 °C, 100 μl of extraction buffer [20% sodium dodecyl sulfate dissolved in a solution of 50% dimethylformamide and 50% deuterium-depleted water (pH 4.7)] was added to each well, and the plates were incubated again at 37 °C for 30 min. Finally, the color intensity was measured using an enzyme-linked immunosorbent assay reader at 570 nm. The presented results are the mean of three independent experiments. Mammalian Cell Viability Analysis. 3T3 mouse fibroblasts and HeLa cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin, and 2 mmol/L 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-well plate and kept overnight for attachment at 37 °C in a humidified atmosphere containing 5% CO2. Quadruplet inserts coated on one side with the nanoassemblyincorporated resin composite restoratives were placed into the wells containing the adhered cells, followed by a 15 h 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 nonviable cells in each sample. The labeled 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% nanoassembly-amalgamated restoratives or the control (0%) restorative. The 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.
■
■
ACKNOWLEDGMENTS
■
REFERENCES
This work is dedicated to the memory of the remarkable Dr. Anszel Sznajder, DMD. The authors 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. Alex Barbul for his assistance with confocal microscopy; Dr. Vered Holdengreber for her assistance in the preparation of bacterial samples for electron microscopy; and Dr. Sigal Rencus-Lazar for her assistance in manuscript editing. They also 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 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.
(1) Zhang, S. Fabrication of Novel Biomaterials through Molecular Self-Assembly. Nat. Biotechnol. 2003, 1171−1178. (2) Ulijn, R. V.; Smith, A. M. Designing Peptide Based Nanomaterials. Chem. Soc. Rev. 2008, 37, 664. (3) Matson, J. B.; Zha, R. H.; Stupp, S. I. Peptide Self-Assembly for Crafting Functional Biological Materials. Curr. Opin. Solid State Mater. Sci. 2011, 15, 225−235. (4) Lakshmanan, A.; Zhang, S.; Hauser, C. A. E. Short SelfAssembling Peptides as Building Blocks for Modern Nanodevices. Trends Biotechnol. 2012, 30, 155−165. (5) Chan, K. H.; Lee, W. H.; Zhuo, S.; Ni, M. Harnessing Supramolecular Peptide Nanotechnology in Biomedical Applications. Int. J. Nanomed. 2017, 12, 1171−1182. (6) Branco, M. C.; Schneider, J. P. Self-Assembling Materials for Therapeutic Delivery. Acta Biomater. 2009, 5, 817−831. (7) Bysell, H.; Månsson, R.; Hansson, P.; Malmsten, M. Microgels and Microcapsules in Peptide and Protein Drug Delivery. Adv. Drug Delivery Rev. 2011, 63, 1172−1185. (8) Habibi, N.; Kamaly, N.; Memic, A.; Shafiee, H. Self-Assembled Peptide-Based Nanostructures: Smart Nanomaterials toward Targeted Drug Delivery. Nano Today 2016, 11, 41−60. (9) Pal, H. A.; Mohapatra, S.; Gupta, V.; Ghosh, S.; Verma, S. SelfAssembling Soft Structures for Intracellular NO Release and Promotion of Neurite Outgrowth. Chem. Sci. 2017, 8, 6171−6175. (10) Roth-Konforti, M. E.; Comune, M.; Halperin-Sternfeld, M.; Grigoriants, I.; Shabat, D.; Adler-Abramovich, L. UV Light− Responsive Peptide-Based Supramolecular Hydrogel for Controlled Drug Delivery. Macromol. Rapid Commun. 2018, 39, No. 1800588. (11) Gelain, F.; Horii, A.; Zhang, S. Designer Self-Assembling Peptide Scaffolds for 3-D Tissue Cell Cultures and Regenerative Medicine. Macromol. Biosci. 2007, 7, 544−551. (12) Maude, S.; Ingham, E.; Aggeli, A. Biomimetic Self-Assembling Peptides as Scaffolds for Soft Tissue Engineering. Nanomedicine 2013, 8, 823−847. (13) Thompson, C. B.; Korley, L. T. J. Harnessing Supramolecular and Peptidic Self-Assembly for the Construction of Reinforced Polymeric Tissue Scaffolds. Bioconjugate Chem. 2017, 28, 1325−1339. (14) Ghosh, M.; Halperin-Sternfeld, M.; Grigoriants, I.; Lee, J.; Nam, K. T.; Adler-Abramovich, L. Arginine-Presenting Peptide Hydrogels Decorated with Hydroxyapatite as Biomimetic Scaffolds for Bone Regeneration. Biomacromolecules 2017, 18, 3541−3550. (15) Wu, E. C.; Zhang, S.; Hauser, C. A. E. Self-Assembling Peptides as Cell-Interactive Scaffolds. Adv. Funct. Mater. 2012, 22, 456−468. (16) Inostroza-Brito, K. E.; Collin, E.; Siton-Mendelson, O.; Smith, K. H.; Monge-Marcet, A.; Ferreira, D. S.; Rodríguez, R. P.; Alonso, M.; Rodríguez-Cabello, J. C.; Reis, R. L.; et al. Co-Assembly,
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b02839.
■
Research Article
Membrane permeation analysis; homogeneous incorporation of the Fmoc-F5-Phe nanoassemblies within the resin composite restorative; stability study of the antibacterial resin composite restorative (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Moumita Ghosh: 0000-0002-8049-7330 Lihi Adler-Abramovich: 0000-0003-3433-0625 Notes
The authors declare no competing financial interest. 21340
DOI: 10.1021/acsami.9b02839 ACS Appl. Mater. Interfaces 2019, 11, 21334−21342
Research Article
ACS Applied Materials & Interfaces Spatiotemporal Control and Morphogenesis of a Hybrid ProteinPeptide System. Nat. Chem. 2015, 7, 897−904. (17) Aviv, M.; Halperin-Sternfeld, M.; Grigoriants, I.; Buzhansky, L.; Mironi-Harpaz, I.; Seliktar, D.; Einav, S.; Nevo, Z.; Adler-Abramovich, L. Improving the Mechanical Rigidity of Hyaluronic Acid by Integration of a Supramolecular Peptide Matrix. ACS Appl. Mater. Interfaces 2018, 10, 41883−41891. (18) McCloskey, A.; Gilmore, B.; Laverty, G. Evolution of Antimicrobial Peptides to Self-Assembled Peptides for Biomaterial Applications. Pathogens 2014, 3, 791−821. (19) Fernandez-Lopez, S.; Kim, H. S.; Choi, E. C.; Delgado, M.; Granja, J. R.; Khasanov, A.; Kraehenbuehl, K.; Long, G.; Weinberger, D. A.; Wilcoxen, K. M.; et al. Antibacterial Agents Based on the Cyclic D,L-Alpha-Peptide Architecture. Nature 2001, 412, 452−455. (20) Rodrigues De Almeida, N.; Han, Y.; Perez, J.; Kirkpatrick, S.; Wang, Y.; Sheridan, M. C. Design, Synthesis, and NanostructureDependent Antibacterial Activity of Cationic Peptide Amphiphiles. ACS Appl. Mater. Interfaces 2019, 11, 2790−2801. (21) Pazos, E.; Sleep, E.; Rubert Pérez, C. M.; Lee, S. S.; Tantakitti, F.; Stupp, S. I. Nucleation and Growth of Ordered Arrays of Silver Nanoparticles on Peptide Nanofibers: Hybrid Nanostructures with Antimicrobial Properties. J. Am. Chem. Soc. 2016, 138, 5507−5510. (22) Qi, R.; Zhou, L.; Zhang, N.; Zhou, C.; Wang, S.; Wang, Y.; Liu, J.; Han, Y.; Zhang, P. Peptide Amphiphiles with Distinct Supramolecular Nanostructures for Controlled Antibacterial Activities. ACS Appl. Bio Mater. 2018, 1, 21−26. (23) Xu, D.; Jiang, L.; Singh, A.; Dustin, D.; Yang, M.; Liu, L.; Lund, R.; Sellati, T. J.; Dong, H. Designed Supramolecular Filamentous Peptides: Balance of Nanostructure, Cytotoxicity and Antimicrobial Activity. Chem. Commun. 2015, 51, 1289−1292. (24) Schnaider, L.; Brahmachari, S.; Schmidt, N. W.; Mensa, B.; Shaham-Niv, S.; Bychenko, D.; Adler-Abramovich, L.; Shimon, L. J. W.; Kolusheva, S.; Degrado, W. F.; et al. Self-Assembling Dipeptide Antibacterial Nanostructures with Membrane Disrupting Activity. Nat. Commun. 2017, 8, No. 1365. (25) Porter, S. L.; Coulter, S. M.; Pentlavalli, S.; Thompson, T. P.; Laverty, G. Self-Assembling Diphenylalanine Peptide Nanotubes Selectively Eradicate Bacterial Biofilm Infection. Acta Biomater. 2018, 77, 96−105. (26) McCloskey, A. P.; Draper, E. R.; Gilmore, B. F.; Laverty, G. Ultrashort Self-Assembling Fmoc-Peptide Gelators for Anti-Infective Biomaterial Applications. J. Pept. Sci. 2017, 23, 131−140. (27) Gahane, A. Y.; Ranjan, P.; Singh, V.; Sharma, R. K.; Sinha, N.; Sharma, M.; Chaudhry, R.; Thakur, A. K. Fmoc-Phenylalanine Displays Antibacterial Activity against Gram-Positive Bacteria in Gel and Solution Phases. Soft Matter 2018, 14, 2234−2244. (28) Ye, Z.; Zhu, X.; Acosta, S.; Kumar, D.; Sang, T.; Aparicio, C. Self-Assembly Dynamics and Antimicrobial Activity of All l- and dAmino Acid Enantiomers of a Designer Peptide. Nanoscale 2019, 11, 266−275. (29) Salick, D. A.; Kretsinger, J. K.; Pochan, D. J.; Schneider, J. P. Inherent Antibacterial Activity of a Peptide-Based β-Hairpin Hydrogel. J. Am. Chem. Soc. 2007, 129, 14793−14799. (30) Liu, L.; Xu, K.; Wang, H.; Tan, P. K. J.; Fan, W.; Venkatraman, S. S.; Li, L.; Yang, Y.-Y. Self-Assembled Cationic Peptide Nanoparticles as an Efficient Antimicrobial Agent. Nat. Nanotechnol. 2009, 4, 457−463. (31) Tripathi, J. K.; Pal, S.; Awasthi, B.; Kumar, A.; Tandon, A.; Mitra, K.; Chattopadhyay, N.; Ghosh, J. K. Variants of SelfAssembling Peptide, KLD-12 That Show Both Rapid Fracture Healing and Antimicrobial Properties. Biomaterials 2015, 56, 92−103. (32) Hughes, M.; Debnath, S.; Knapp, C. W.; Ulijn, R. V.; et al. Antimicrobial Properties of Enzymatically Triggered Self-Assembling Aromatic Peptide Amphiphiles. Biomater. Sci. 2013, 1, 1138. (33) Laverty, G.; McCloskey, A. P.; Gilmore, B. F.; Jones, D. S.; Zhou, J.; Xu, B. Ultrashort Cationic Naphthalene-Derived SelfAssembled Peptides as Antimicrobial Nanomaterials. Biomacromolecules 2014, 15, 3429−3439.
(34) Hu, B.; Shen, Y.; Adamcik, J.; Fischer, P.; Schneider, M.; Loessner, M. J.; Mezzenga, R. Polyphenol-Binding Amyloid Fibrils Self-Assemble into Reversible Hydrogels with Antibacterial Activity. ACS Nano 2018, 12, 3385−3396. (35) Maity, S.; Nir, S.; Zada, T.; Reches, M. Self-Assembly of a Tripeptide into a Functional Coating That Resists Fouling. Chem. Commun. 2014, 50, 11154−11157. (36) Azevedo, H. S.; Falanga, A.; Galdiero, S.; Franci, G.; Lombardi, L.; Shi, Y.; Galdiero, E.; Chourpa, I.; de Alteriis, E. Enhancing the Potency of Antimicrobial Peptides through Molecular Engineering and Self-Assembly. Biomacromolecules 2019, 20, 1362−1374. (37) Du, X.; Zhou, J.; Shi, J.; Xu, B. Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials. Chem. Rev. 2015, 115, 13165−13307. (38) Tao, K.; Levin, A.; Adler-Abramovich, L.; Gazit, E. FmocModified Amino Acids and Short Peptides: Simple Bio-Inspired Building Blocks for the Fabrication of Functional Materials. Chem. Soc. Rev. 2016, 45, 3935−3953. (39) Burch, R. M.; Weitzberg, M.; Blok, N.; Muhlhauser, R.; Martin, D.; Farmer, S. G.; Bator, J. M.; Connor, J. R.; Green, M.; Ko, C. N(Fluorenyl-9-Methoxycarbonyl) Amino Acids, a Class of Antiinflammatory Agents with a Different Mechanism of Action. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 355−359. (40) Debnath, S.; Shome, A.; Das, D.; Das, P. K. Hydrogelation Through Self-Assembly of Fmoc-Peptide Functionalized Cationic Amphiphiles: Potent Antibacterial Agent. J. Phys. Chem. B 2010, 114, 4407−4415. (41) Ryan, D. M.; Anderson, S. B.; Senguen, F. T.; Youngman, R. E.; Nilsson, B. L. Self-Assembly and Hydrogelation Promoted by F5 -Phenylalanine. Soft Matter 2010, 6, 475−479. (42) Rajbhandary, A.; Brennessel, W. W.; Nilsson, B. L. Comparison of the Self-Assembly Behavior of Fmoc-Phenylalanine and Corresponding Peptoid Derivatives. Cryst. Growth Des. 2018, 18, 623−632. (43) Halperin-Sternfeld, M.; Ghosh, M.; Sevostianov, R.; Grigoriants, I.; Adler-Abramovich, L. Molecular Co-Assembly as a Strategy for Synergistic Improvement of the Mechanical Properties of Hydrogels. Chem. Commun. 2017, 53, 9586−9589. (44) World Health Organization. Antimicrobial Resistance: Global Report on Surveillance; WHO, 2014; p 8. (45) Bagramian, R. A.; Garcia-Godoy, F.; Volpe, A. R. The Global Increase in Dental Caries. A Pending Public Health Crisis. Am. J. Dent. 2009, 22, 3−8. (46) Righolt, A. J.; Jevdjevic, M.; Marcenes, W.; Listl, S. Global-, Regional-, and Country-Level Economic Impacts of Dental Diseases in 2015. J. Dent. Res. 2018, 97, 501. (47) Kassebaum, N. J.; Bernabé, E.; Dahiya, M.; Bhandari, B.; Murray, C. J. L.; Marcenes, W. Global Burden of Untreated Caries: A Systematic Review and Metaregression. J. Dent. Res. 2015, 94, 650− 658. (48) Kermanshahi, S.; Santerre, J. P.; Cvitkovitch, D. G.; Finer, Y. Biodegradation of Resin-Dentin Interfaces Increases Bacterial Microleakage. J. Dent. Res. 2010, 89, 996−1001. (49) Bourbia, M.; Ma, D.; Cvitkovitch, D. G.; Santerre, J. P.; Finer, Y. Cariogenic Bacteria Degrade Dental Resin Composites and Adhesives. J. Dent. Res. 2013, 92, 989−994. (50) Cocco, A. R.; de Oliveira da Rosa, W. L.; da Silva, A. F.; Lund, R. G.; Piva, E. A Systematic Review about Antibacterial Monomers Used in Dental Adhesive Systems: Current Status and Further Prospects. Dent. Mater. 2015, 31, 1345−1362. (51) Imazato, S.; Ma, S.; Chen, J. H.; Xu, H. H. K. Therapeutic Polymers for Dental Adhesives: Loading Resins with Bio-Active Components. Dent. Mater. 2014, 30, 97−104. (52) Melo, M. A. S.; Guedes, S. F. F.; Xu, H. H. K.; Rodrigues, L. K. A. Nanotechnology-Based Restorative Materials for Dental Caries Management. Trends Biotechnol. 2013, 459−467. (53) Beyth, N.; Farah, S.; Domb, A. J.; Weiss, E. I. Antibacterial Dental Resin Composites. React. Funct. Polym. 2014, 75, 81−88. (54) Beyth, N.; Yudovin-Farber, I.; Basu, A.; Weiss, E. I.; Domb, A. J. Antimicrobial Nanoparticles in Restorative Composites. In Emerging 21341
DOI: 10.1021/acsami.9b02839 ACS Appl. Mater. Interfaces 2019, 11, 21334−21342
Research Article
ACS Applied Materials & Interfaces Nanotechnologies in Dentistry, 2nd ed.; William Andrew Publishers, 2018; pp 41−58. (55) Makovitzki, A.; Avrahami, D.; Shai, Y. Ultrashort Antibacterial and Antifungal Lipopeptides. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15997−16002. (56) Matalon, S.; Weiss, E. I.; Gorfil, C.; Noy, D.; Slutzky, H. In Vitro Antibacterial Evaluation of Flowable Restorative Materials. Quintessence Int. 2009, 40, 327−332. (57) Weiss, E. I.; Shalhav, M.; Fuss, Z. Assessment of Antibacterial Activity of Endodontic Sealers by a Direct Contact Test. Endod. Dent. Traumatol. 1996, 12, 179−184. (58) Zilberman, M.; Elsner, J. J. Antibiotic-Eluting Medical Devices for Various Applications. J. Controlled Release 2008, 130, 202−215. (59) Black, J. Biological Performance of Materials: Fundamentals of Biocompatibility, 4th ed.; Taylor & Francis, 2005. (60) Roydhouse, R. H. Punch-Shear Test for Dental Purposes. J. Dent. Res. 1970, 49, 131−136. (61) Mount, G. J.; Makinson, O. F.; Peters, M. C. R. B. The Strength of Auto-Cured and Light-Cured Materials. The Shear Punch Test. Aust. Dent. J. 1996, 41, 118−123. (62) Nomoto, R.; Carrick, T. E.; McCabe, J. F. Suitability of a Shear Punch Test for Dental Restorative Materials. Dent. Mater. 2001, 17, 415−421. (63) Pilo, R.; Ben-Amar, A.; Barnea, A.; Blasbalg, Y.; Levartovsky, S. The Effect of Resin Coating on the Shear Punch Strength of Restorative Glass Ionomer Cements. Clin. Oral Invest. 2017, 21, 1079−1086.
21342
DOI: 10.1021/acsami.9b02839 ACS Appl. Mater. Interfaces 2019, 11, 21334−21342
