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Multifunctional Mesoporous Carbon Capsules and their Robust Coatings for Encapsulation of Actives: Antimicrobial and Antibioadhesion Functions Gargi Mishra,† Nitesh Mittal,†,‡ and Ashutosh Sharma* Department of Chemical Engineering and Centre of Nanosciences, Indian Institute of Technology Kanpur, Kanpur-208016, India S Supporting Information *

ABSTRACT: We present the synthesis and applications of multifunctional hollow porous carbon spheres with wellordered pore architecture and ability to encapsulate functional nanoparticles. In the present work, the applications of hollow mesoporous carbon capsules (HMCCs) are illustrated in two different contexts. In the first approach, the hollow capsule core is used to encapsulate silver nanoparticles to impart antimicrobial characteristics. It is shown that silver-loaded HMCCs (concentration ∼100 μg/mL) inhibit the growth and multiplication of bacterial colonies of Escherichia coli (Gramnegative) and Staphylococcus aureus (Gram-positive) up to 96% and 83%, respectively. In the second part, the fabrication of hierarchical micro- and nanostructured superhydrophobic coatings of HMCCs (without encapsulation with silver nanoparticles) is evaluated for anti-bioadhesion properties. Studies of protein adsorption and microorganism and platelet adhesion have shown a significant reduction (up to 100%) for the HMCCbased superhydrophobic surfaces compared with the control surfaces. Therefore, this unique architecture of HMCCs and their coatings with the ability to encapsulate functional materials make them a promising candidate for a variety of applications. KEYWORDS: mesoporous carbon capsules, silver encapsulation, antimicrobial, superhydrophobic, anti-bioadhesion

1. INTRODUCTION Mesoporous carbon capsules (MCCs) have drawn considerable attention for their remarkable features like ordered porous architecture, a high specific surface area with large pore volume, and their ability to encapsulate inorganic materials into the hollow core of mesostructured carbon shells.1 Different nanoparticles can be incorporated within mesoporous carbon particles to create a core/shell architecture without bringing down the mesostructure.2 Compared with the traditional methods of doping carbon nanoparticles with inorganic nanomaterials, hollow mesoporous carbon capsules (HMCCs) act as reservoirs to store molecules that are slowly released, which is well-demonstrated for their biocompatibility and drug delivery applications.2−6 Elemental silver and silver salts have been known for several years as antimicrobial agents for healthcare applications.7 The antimicrobial activity of Ag is dependent on Ag cation (Ag+), which tends to bind strongly to electron-donating groups present in biomolecules.8,9 Researchers have tried different methods to deposit metallic Ag onto various polymeric substrates for the release of Ag in a pathogenic environment.10−12 Attempts have also been made to encapsulate Ag+ ions or crystals into the nanoparticles to effectively control the diffusion between the Ag+ ions and the contaminated medium containing bacteria for enhanced antimicrobial activities.13 © XXXX American Chemical Society

HMCCs with the ability to encapsulate functional materials into the core and good dispersibility in water can be utilized toward successful treatment of wastewater, where antimicrobial activities are required.14−16 Herein we describe the in situ synthesis of Ag nanoparticles encapsulated in MCCs with a hollow core and testing of the antimicrobial effectiveness of the materials for both Gram-negative (GN) and Gram-positive (GP) bacteria. The bacteria used in the current study are Escherichia coli as the GN model and Staphylococcus aureus as the GP model. Additionally, for biomaterials synthesis, controlling the adsorption of proteins and subsequent adhesion of cells over the surfaces has been a key issue, as it directs the architecture and performance of the tissue.17−19 Several implants used in the body require a coating to minimize adsorption of proteins and cells. Nevertheless, deployment of these implants may stimulate foreign body reactions and lead to blood coagulation.20−23 Exclusively, many times the implanted surfaces provide a suitable platform for adhesion of microbes, leading to infection and implant failure.24−26 To reduce and avoid the incidences of Special Issue: Focus on India Received: June 28, 2016 Accepted: October 12, 2016

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DOI: 10.1021/acsami.6b07831 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces these complications, a variety of coatings and compositions have been proposed.27−29 However, it is well-known that the adsorption of proteins and adhesion of microorganisms and platelets onto a solid substrate are highly dependent on the wetting properties and topography of the surface.30−33 As a result, superhydrophobic surfaces have emerged as potential alternatives to achieve antifouling properties.34−38 Another appealing facet of HMCCs, besides their ability to encapsulate functional materials in the hollow core, is their unique architecture that allows fabrication of hierarchical superhydrophobic coatings. We have recently demonstrated the idea of fabricating superhydrophobic surfaces using HMCCs.39 In contrast to the complicated techniques to achieve superhydrophobicity that are very specific to surface morphologies, these coatings can be applied conformably to any surface via a simple brush-on-process irrespective of the surface type and morphology. The coatings have demonstrated good chemical resistance and mechanical durability. Here we explore the response of HMCC-based superhydrophobic coatings to provide defense against the adhesion of proteins, bacteria, mammalian cells, and platelets, even without encapsulation and release of actives. Such a baseline also establishes the potential multifunctional use of HMCC coatings with encapsulation of actives, including Ag nanoparticles.

Figure 1. (a) TEM micrograph of MCCs with a hollow core. (b) HRTEM micrograph of MCCs encapsulated with Ag nanoparticles in the core. The scale bars in (a) and (b) are 200 nm. (c) Zoomed view in which Ag nanoparticles with sizes of 8−14 nm are visible. The scale bar is 20 nm. XRD patterns of HMCCs and Ag@MCC to confirm the presence of Ag nanoparticles encapsulated in the HMCCs are shown in Figure S1 in the Supporting Information. (d) Nitrogen adsorption/ desorption isotherm and (inset) pore size distribution of the HMCCs.

2. EXPERIMENTAL SECTION 2.1. Materials. Sodium borohydride (NaBH4), sodium bicarbonate (NaHCO3), sodium chloride (NaCl), silver nitrate (AgNO3), yeast extract powder, bacterial tryptone, agar, formaldehyde, phosphatebuffered saline (PBS), ethanol, and dimethyl sulfoxide (DMSO) were obtained from Merck Chemicals, India. Fluorescein diacetate (FDA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), rhodamine isothiocyanate (RITC), bovine serum albumin (BSA), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin−streptomycin, trypsin−EDTA, and Luria−Bertani (LB) agar plates were obtained from Sigma-Aldrich, India. The bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Scientific, India. The above chemicals were used as received. HeLa and L929 cell lines were purchased from National Centre for Cell Science, Pune, India. E. coli and S. aureus were purchased from the American Type Culture Collection (ATCC). Human platelet-rich plasma (PRP) was obtained from the Pathology Department of G.S.V.M. Medical College, Kanpur, India. Smooth stainless steel (SS) substrates with dimensions of 1.5 cm × 1.5 cm were used as controls throughout this study. 2.2. Preparation of Hollow Mesoporous Carbon Capsules. Nanometer-sized carbon capsules with a hollow core and mesoporous shell (Figure 1a) were prepared by a protocol similar to that reported by Mittal et al.40 2.3. Synthesis of Silver-Encapsulated Mesoporous Carbon Capsules. HMCCs (10 mg) were dispersed in AgNO3 solution (1 mL, 0.1 M) by sonication for 30 min and incubated for 15 min. Then an aqueous solution of NaBH4 (100 μL, 0.1 M) was added, and the mixture was allowed to stir for 25 min. The Ag-nanoparticle-loaded carbon capsules (Ag@MCC) (Figure 1b) were separated by centrifugation at 6500 rpm. The Ag@MCC were washed with water to remove excess unencapsulated Ag nanoparticles and reactants. The above-mentioned protocol leads to the in situ synthesis of colloidal Ag into the porous and hollow thin-shelled MCCs. By this procedure, colloidal Ag nanoparticles were synthesized by reduction of AgNO3 by adding an excess of the reducing agent NaBH4 through following chemical reaction: AgNO3 + NaBH4 → Ag +

2.4. Fabrication of Superhydrophobic Surfaces. HMCC-based superhydrophobic surfaces were prepared by a procedure similar to that described in our previous study on MCC-based coatings with multiple functionalities.39 In brief, HMCCs with sizes of ∼200−300 nm were dispersed in poly(vinylidene fluoride) (PVDF) dissolved in N-methyl-2-pyrrolidone (NMP). Coatings were applied on the surface of SS substrates with a paint brush to achieve the superhydrophobicity. 2.5. Contact Angle Measurements. Static contact angles (CAs) and dynamic sliding angles (SAs) were measured on a DataPhysics contact angle goniometer (model OCA 35) at ambient temperature. CAs were measured by placing 4 μL droplets of deionized (DI) water onto the substrates, and 12 μL droplets were used for the SA measurements. 2.6. Characterization. HMCCs and Ag@MCC were characterized by transmission electron microscopy (TEM) (FEI Techai G2 U-Twin microscope) at an accelerating voltage of 200 kV and high-resolution TEM (HR-TEM) (FEI Titan G2 60-300 microscope), respectively. HMCC-based superhydrophobic surfaces were characterized by fieldemission scanning electron microscopy (FESEM) (SUPRA 40 VP Gemini microscope, Zeiss, Oberkochen, Germany) at an accelerating voltage of 10 kV. X-ray diffraction (XRD) was performed on a Thermo Electron diffractometer operating in the θ−2θ Bragg configuration using Cu Kα radiation (λ = 1.506 Å) at 40 kV from 20° to 80° at a scan rate of 2° min−1 in order to understand the crystalline nature of the HMCCs and Ag@MCC. FTIR spectra for the superhydrophobic surface were recorded on a Bruker Tensor spectrometer over the frequency range of 800−1600 cm−1. The zeta potential of the HMCCbased coatings was measured with a Delsa Nano submicron particle size and zeta potential particle analyzer (Beckman Coulter) at 25 °C. The MCC pore size distribution (Figure 1d inset) was measured using a Brunauer−Emmett−Teller (BET) surface area analyzer (Quantachrome Autosorb iQ). The pore size distribution was analyzed utilizing the Barrett−Joyner−Halenda (BJH) method.42 2.7. Microbial Culture. E. coli (GN) and S. aureus (GP) bacteria were cultured in LB broth at 37 °C. Bacterial cultures were obtained from already-plated LB agar plates (Sigma-Aldrich) stored at 37 °C.

1 1 H 2 + B2H6 + NaNO3 2 2

The method used in this procedure produced Ag nanoparticles that in general were 8−14 nm in size.41 B


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Figure 2. (a, b) Agar plate assays to check the antibacterial activity of Ag@MCC at different concentrations (μg/mL) on (a) E. coli and (b) S. aureus. (c, d) Growth curves of (c) E. coli and (d) S. aureus in the presence of different concentrations (0−100 μg/mL) of Ag@MCC. (e, f) Plots of the number of colonies of (e) E. coli and (f) S. aureus vs the concentration of Ag@MCC. Single colonies were picked up from the agar plates and inoculated in LB broth overnight at 37 °C. From this, 100 μL of culture was used to inoculate fresh LB broth, which was then cultured for 3 h. A 500 μL aliquot of bacterial culture was taken from it and diluted to get an optical density (OD) of 0.5 at 600 nm. 2.8. Antimicrobial Tests. Sterilized LB medium was inoculated with 500 μL of a cultured bacterial suspension of E. coli or S. aureus, followed by the addition of Ag@MCC (0, 25, 50, or 100 μg/mL) into the suspension culture and incubation for various times (0.5−24 h) in an incubator−shaker at 37 °C and 50 rpm. The OD of the suspension culture was measured at various time points (0.5−24 h) using a UV− vis spectrophotometer (Multiskan Spectrum, Thermo Scientific). The bacterial growth kinetics was measured by observing the OD at 600 nm at different time points, as reported in the literature.43,44 After 24 h of incubation, 100 μL of culture medium was taken out and serially diluted (1/104) to give a very dilute culture of bacteria, which was plated onto solidified LB agar plates and incubated at 37 °C for 24 h.

After 24 h, the number of bacterial colonies on the plates was counted and plotted against the concentration of Ag@MCC. The experiment was repeated thrice, and all of the readings were taken in triplicate. 2.9. Protein Adhesion Test. BSA protein was labeled with RITC to make it visible under the fluorescence microscope. BSA (2 mg/mL in 4% NaHCO3 solution) was incubated with 50 μL of a freshly prepared solution of RITC (1 mg/mL) under constant stirring for 2−4 h in a dark room. RITC−BSA was separated from RITC using a PD10 column in PBS. This RITC-labeled BSA (500 μg/mL) was incubated with the HMCC-coated SS substrates (1.5 cm × 1.5 cm) in a dark room and allowed to adsorb on the substrate for 24 h. After 24 h, the substrates were taken out and washed with PBS to remove loosely bound and free protein moieties. The obtained samples were observed under a fluorescence microscope, and images were taken to get the qualitative estimation of protein adsorption. Protein adsorption tests were done on the substrates using BSA protein solution in PBS (50 μg/mL). Substrates were incubated with BSA solution at 37 °C for C


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Figure 3. (a) FESEM image showing hierarchical roughness obtained by the HMCC-based superhydrophobic coating on a stainless steel surface (the scale bar is 10 μm). The inset shows a magnified view (the scale bar is 200 nm). (b) Shape of a DI water droplet on the SS surface coated with HMCCs. (c) FTIR spectrum of a HMCC-based superhydrophobic surface. 24 h, after which the protein solutions over the substrates were taken out and quantified for the protein concentration using the BCA assay. The amount of protein adsorbed on the substrate was calculated by subtracting the calculated concentration from the initial concentration of protein used for incubation of the substrates. 2.10. Bacterial Adhesion Test. S. aureus bacteria were cultured in sterilized LB culture medium and incubated for 8 h at 37 °C in a shaking incubator. HMCC-coated superhydrophobic substrates were sterilized and submerged in 2 mL of LB medium. The medium was then inoculated with 50 μL of bacterial culture suspension. At different time points, the HMCC-coated substrates were taken out washed with PBS twice to remove the unadhered bacteria. The samples were analyzed for adhered bacteria on the surface. The quantification was done using the FDA assay at different time points. Samples were collected at various time points, and FDA stain (10 μg/mL, 1 mL) was added to each substrate, followed by incubation for 30 min. After 30 min, the supernatant was collected, and the OD at 488 nm was measured. The OD values were plotted against time. For microscopic evaluation, substrate samples after incubation in bacterial suspension were washed with PBS multiple times, fixed with 2.5% glutaraldehyde, and washed with PBS again. Substrates were then dried using an ethanol gradient (10−100%) each for 10 min and dried and observed by FESEM. 2.11. Mammalian Cell Adhesion and in Vitro Biocompatibility Tests. Uncoated and HMCC-coated SS substrates were autoclaved and kept in 24-well tissue culture plates. HeLa and L929 cells were cultured in DMEM to achieve 70% confluency in a T-25 cm2 flask, and cells were harvested after trypsinization. Then 105 cells were seeded in each well, and 1 mL of DMEM supplemented with 10% FBS and 1% penicillin−streptomycin was added to each well. Tissue culture plates were incubated in a CO2 incubator at 37 °C. Substrates were taken out at different times, fixed with 4% formaldehyde solution, and dehydrated using different ethanol concentrations (10−100%) by dipping the substrates in ethanol for 10 min. After dehydration, the samples were coated with gold for 10 s using a gold sputterer and observed by FESEM. Quantitative estimation of the number of cells adhered to the substrates was performed by measuring the cell viability using the MTT assay. Cells (HeLa and L929) were incubated with substrates for various times (1−7 days), and for each time point the cell viability was measured using MTT dye, which measures the metabolic activity of the viable cells. At various time points, medium from each of the wells was discarded, and MTT dye (0.5 mg/mL, 1 mL) was added to each well. After 4 h, the MTT dye was discarded, and DMSO (1 mL) was added to each well to form a purple complex solution whose OD was measured at 560 nm. Cell numbers were then calculated from the OD values using a standard curve of number of cells plotted versus OD of the purple complex solution formed in the MTT viability assay for each cell line (HeLa or L929). 2.12. Platelet Adhesion Test. Uncoated and coated substrates were equilibrated with PBS for 6 h, and 3 mL of PRP after dilution

(1:100) in citrate buffer was added to each substrate. The platelets were allowed to adhere to the substrates for 3 h, after which the samples were washed with PBS and fixed with glutaraldehyde (2.5% v/ v). Thereafter substrates were dehydrated using different ethanol concentrations (10−100%) by dipping the substrates in ethanol for 10 min and then observed by FESEM.

3. RESULTS AND DISCUSSION 3.1. Antimicrobial Testing of Silver-Loaded Carbon Capsules. HMCCs have been shown to act as reservoirs of various nanoparticles.2 Using the same approach, we encapsulated in situ Ag nanoparticles inside HMCCs to be used as carriers to impart antimicrobial activities to a pathogenic medium, so that it may limit the possibilities of bacterial infection. Bare carbon capsules with pore sizes of 3−4 nm were used to encapsulate the Ag nanoparticles (Figure 1a−c). We checked the effect of antimicrobial activities by using a UV−vis spectrophotometer to read the optical densities at 600 nm of the bacterial suspensions incubated with various concentrations (25−100 μg/mL) of Ag-nanoparticle-loaded carbon capsules for 24 h (Figure 2a,b). Then growth curves were plotted (Figure 2c,d), and an agar plate assay was also done to find out the number of colonies (Figure 2e,f). We observed that Ag@MCC at a concentration of 100 μg/ mL was able to effectively inhibit the growth of both GN and GP bacteria. It can be seen from the agar plate assay that for E. coli, Ag@MCC at 25 μg/mL causes an approximately 90% reduction in the number of colonies compared with the control experiment (Figure 2e). Higher concentrations of 50 μg/mL and 100 μg/mL are able to cause even better bactericidal effects, reaching reductions of around 95% in the number of colonies. Similar kind of bactericidal effects are seen against S. aureus, where a reduction of more than 80% is seen in the bacterial colonies at a concentration of 100 μg/mL (Figure 2f). The bactericidal effect is more pronounced in E. coli compared with S. aureus because of the difference in the cell walls of GN and GP bacteria. It has already been reported that E. coli shows more sensitivity toward Ag+ ions than S. aureus because of the presence of a higher percentage of peptidoglycans in the cell wall of the former compared with the latter.45 The decrease in bacterial number in LB broth and the agar plate assay can be attributed to the antibacterial action of Ag+ ions and nanoparticles present in the core and also on the shells of the carbon capsules. In addition, the surface of the carbon capsules might also provide surface area for interaction of bacteria and Ag nanoparticles, depending on the surface characteristics of the nanoparticles and the cytotoxicity.13 The D


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Figure 4. Protein adsorption on the uncoated SS substrate and the SS surface coated with HMCCs. (a, b) Fluorescence microscopy images of the protein adsorption on (a) uncoated and (b) HMCC-coated SS surfaces (scale bars are 100 μm). (c) Quantitative estimation and comparison of protein adsorption on the bare SS surface and the SS surface coated with HMCCs.

of 66 kDa. Qualitatively, as can be observed in Figure 4b, the surface is very clean for a superhydrophobic HMCC surface, as only a few red dots, which show the presence of protein over the surface, are visible. In contrast, in the case of a smooth SS substrate, the red dots are present over the whole surface (Figure 4a). Protein adhesion was quantified using the BCA assay to find out the amount of protein adhered per square centimeter of coating. The amount of protein adsorbed on the coated HMCC substrate was ∼3.5 pg/cm2, versus ∼27 pg/cm2 for the uncoated SS substrate, corresponding to an 87% reduction in the protein adsorption on the coated surface compared with the control surface. In contrast to HMCC coating, there are various other protein antifouling approaches available, such as attaching PEG chains on the substrates,50 tethering proteases on the substrates, self-assembled monolayer (SAM)-based surfaces,51 and zwitterionic surfaces.52 However, most of them have drawbacks in terms of either effective resistivity against proteins or cumbersome fabrication methods.53−55 Among the superhydrophobic coatings, a similar kind of antiprotein adsorption behavior is also shown by silicaparticle-based hierarchical layer-by-layer coatings.56 HMCCbased coatings are a good alternative to the above-mentioned approaches because they are easy to fabricate as well as robust and also provide efficient protein resistance. 3.2.2. Adhesion of Bacteria. HMCC-based coatings have shown reduced bacterial adhesion for the highly pathogenic GN bacteria E. coli in our previous work.39 In the current study, we have exploited the response of the GP bacteria S. aureus toward the adhesion on HMCC-based superhydrophobic surfaces. Results from the incubation of the model implants (uncoated and HMCC-coated SS substrates) in the bacterial suspension culture for a period of 24 h have shown that there is muchreduced adhesion of bacteria (∼92%) on the surface coated with HMCCs compared with the uncoated surface, as shown in Figure 5. The current results are in good agreement with our previous findings on the adhesion of E. coli, where a 91% reduction in the adhesion over MCC-based surfaces was observed.39 The adhesion of bacteria on different substrates was measured using the FDA assay. FDA is a fluorescent dye that stains live bacterial cells. Bacterial adhesion requires a time window that is dissimilar for different bacteria and is dependent on the development of a conditioning layer made up of proteins and organic and inorganic moieties that cover and modify the surface.57,58 A superhydrophobic surface, being repellent to aqueous nutrient media, reduces the possibility to form this conditioning layer. Under in vitro conditions, we overestimate the number of bacteria because they are not

detailed mechanisms associated with the antibacterial activity of Ag+ ions/nanoparticles suspended in a medium are explained in a previous study by Loza and co-workers.46 3.2. Structural Characterization and Anti-bioadhesion of Superhydrophobic Surfaces. The heterogeneous microand nanostructured superhydrophobic surfaces based on HMCCs were analyzed using FESEM (Figure 3a). The hierarchical roughness from HMCCs integrated with the low surface energy of the polymer binder PVDF leads to the superhydrophobicity of the surfaces. In our previous study, we found out that the superhydrophobicity is strongly dependent on the fraction of PVDF relative to HMCCs.39 Thus, under the optimal conditions, the relative amount of PVDF with respect to HMCC was kept at 1 wt % in the finally dried coating deposited over the smooth SS substrates, which were used as reference controls in this study. More detailed information on the surface roughness and characterization is included in our previous work.39 Figure 3b shows the typical image of a DI water droplet on the as-prepared superhydrophobic surface. A static contact angle value of 160.6° with a sliding angle of 5.3° was observed on the HMCC-coated surface, compared with the CA value of 80.7° with no sliding of the droplet on the hydrophilic uncoated SS surface (Figure S2 in the Supporting Information). The chemical nature of the HMCC-coated surface was examined by FTIR spectroscopy. Figure 3c shows the FTIR spectrum of the superhydrophobic surface, where the peaks between 1000 and 1200 cm−1 correspond to the carbon− fluorine bonds, which contributed toward the low surface energy of the surface.47 Furthermore, the universal applicability of the HMCC-based coatings over metals, plastics, glass, and fibers irrespective of the surface morphology of the material with good mechanical durability also contributes toward the versatility of this coating for the synthesis of advanced biomaterials with antifouling properties.39 3.2.1. Adsorption of Proteins. The adsorption of proteins on the substrate marks the beginning of biological interactions on an implant.20 The conditioning film of proteins formed on the substrate begins bacterial and mammalian cell adhesion.48 Protein fouling reduces the sensitivity of in vitro diagnostics assays, reduces the efficacy of biological implants, leads to malfunctioning of the devices, and engenders undesirable side effects.22,49 To overcome these drawbacks, we propose that HMCC coatings can be used on implants to avoid fouling of surfaces. We checked the RITC-labeled BSA protein adsorption on HMCC-coated model substrates and compared it with that on SS substrates (Figure 4). BSA protein adopts a globular shape with a diameter of 12 nm, and it has a molecular weight E


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Figure 6. Testing of HeLa cell adhesion on the uncoated SS surface and the SS surface coated with HMCCs. (a) FESEM image of HeLa cells adhered to the SS surface (scale bar is 100 μm) with a magnified view in the inset (scale bar is 10 μm) after 4 days (96 h) of incubation. (b) FESEM image of the HMCC-coated SS surface (scale bar is 100 μm) with a magnified view in the inset (scale bar is 10 μm). (c) Quantitative estimation and comparison of the numbers of cells adhered on the uncoated and HMCC-coated SS surfaces after 1, 4, and 7 days. It can be seen that larger numbers of HeLa cells adhered to the uncoated SS surface than to the SS surface coated with HMCCs, where very few cells adhered.

Figure 5. Comparison of the adhesion of bacteria on the SS surface (reference) and the SS surface coated with HMCCs after a period of 24 h. (a) FESEM image of the bacterial adhesion on the SS surface. It can be seen that S. aureus adhered and spread out nicely on the surface, leading to the formation of a biofilm. (b) FESEM image of the SS surface coated with HMCCs after the bacterial adhesion experiment. The surface is devoid of any bacterial colony except for a few individual bacteria adhered at a few locations. The scale bars in (a) and (b) are 5 μm. (c) Quantification of adhered bacteria on the uncoated (SS) and HMCC-coated SS surfaces.

after 4 days of culture onto the SS surface. Over the control surface, a significant number of cells can be visualized, which confirms that cells adhered and spread greatly on the surface (Figure 6a). The current findings are consistent with previous work that examined the adhesion of HeLa cells onto the SS surfaces and concluded that cells can adhere to, proliferate on, and differentiate over such surfaces. For the superhydrophobic surface, the number of cells adhered is noticeably lower compared with the control surface (Figure 6b). On the SS surface, HeLa cells formed a confluent two-dimensional (2D) film, as can be seen in Figure 6a, whereas on the HMCC-coated substrates HeLa cells are seen individually at a few spots only (Figure 6b). This could be a result of the fact that the medium suspension containing the cells was not in good contact with the entire surface, as predicted by the Cassie−Baxter model for superhydrophobic surfaces.65 Moreover, after examination of the high-magnification picture of HeLa cell adhesion over the SS surface (Figure 6a) it is possible to comment that the cells are strongly attached to the surface. The quantitative results for cell culture durations of 1, 4, and 7 days are shown in Figure 6c. The density of cells constantly increased on the control SS surface measured on the first, fourth, and seventh days, showing that cells entered the exponential phase of growth with time. In contrast, the density of cells was much lower over the HMCCbased superhydrophobic surface and decreased with time until day 7. The current results suggest that the cells that adhered to the superhydrophobic surfaces initially and then slowly lost their viability because they were not able to spread properly over these reported water-repellant surfaces.65 Similar results were seen with L929 cells, where the SS surface showed 60−70% confluency but the HMCC-based superhydrophobic surface was devoid of any cells (Figure 7). It is noted that L929 cells adhered to the control SS surface in a

subjected to the shear flow of liquid, which can occur inside the blood pool and contribute further toward the reduction of the bacterial adhesion. Moreover, the adhesion of bacteria to a surface is also dependent on the electrostatic interaction between the charge on the bacterial surface and that on the surface against which bacteria are going to adhere.59 In this case, the HMCC-coated surface is found to have a negative value of the zeta potential (−33.58 mV at pH 7), and the surfaces of S. aureus are also known to possess negative zeta potential values in general, which therefore can lead to electrostatic repulsion between the two and possibly contribute toward the reduction in adhesion.60,61 It is noted that the current results are in agreement with antibacterial adhesion reported with negatively charged carbon-based amorphous films and contrary to a study where researchers have reported favorable antimicrobial effects with positively charged surfaces compared with the negatively charged surfaces of S. aureus.59,61 3.2.3. Adhesion of Mammalian Cells. In general, the adhesion of cells is mediated by the molecules or proteins of the cell surfaces.62 Though we observed very minimal protein adsorption on the HMCC-coated substrate (Figure 4b), we cross-checked the antiadhesive property of the substrate for mammalian cells. To investigate the cellular behavior and adhesion of cells on control SS and HMCC-coated superhydrophobic surfaces, HeLa and L929 cells were used as models.63,64 Figure 6 displays the morphology of HeLa cells F


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hydrophobic (coated) (Figure 8b) SS surfaces after 3 h of incubation. It can be seen from Figure 8a that the platelets mask the whole substrate, and the inset image clearly shows that platelets form clustered patches. In contrast, as shown in Figure 8b, the HMCC-coated surface looks devoid of platelets, and visually one can hardly see any platelets adhered to the HMCC-coated substrate. However, no quantitative estimation was done to find out the numbers of platelets adhered to these substrates.

4. CONCLUSIONS We have explored the antimicrobial effect of Ag@MCC on both GN and GP bacteria by the LB agar plate assay. The Ag@ MCC, supplemented with a fairly small amount of Ag, exhibit superior antimicrobial activity. In addition, we have also reported the facile synthesis of HMCC-based superhydrophobic surfaces that work as robust nonfouling surface coatings even without any encapsulated actives. The combination of micro- and nanostructured features from HMCCs with lowsurface-energy fluorinated PVDF resulted in surfaces that reduce the adsorption of protein by 87% compared with the hydrophilic SS substrate. Results from the adhesion of pathogenic bacteria (S. aureus) and mammalian cells (HeLa and L929) also indicate a significant reduction in comparison with the control surfaces. Moreover, results from the reduction in platelet adhesion by almost 100% observed in the current study on HMCC-based superhydrophobic surfaces can also help in largely improving the blood compatibility of biomaterials through the simple introduction of hierarchical structure over the surfaces. We envision that the ability to encapsulate functional nanoparticles into the core together with the ease of achieving superhydrophobicity for nonfouling surface properties makes this class of carbon-based materials excellent candidates as promising biomaterials.

Figure 7. Testing of L929 cell adhesion on the uncoated SS surface and the SS surface coated with HMCCs. (a) FESEM image of L929 cells adhered to the SS surface after 4 days (96 h) of incubation. (b) FESEM image of the HMCC-coated SS surface. The scale bars in (a) and (b) are 50 μm. (c) Quantitative estimation and comparison of the numbers of cells adhered to the uncoated and HMCC-coated SS surfaces after 1, 4, and 7 days.

different manner than for the HeLa cells, where a 2D film was observed. In the case of L929 cells, it was observed that the cells adhered more or less individually over the control SS surface. The shapes of the L929 cells adhered over the SS and superhydrophobic surfaces are in agreement with the previous study on L929 adhesion over flat and patterned surfaces.66,67 3.2.4. Adhesion of Platelets. Platelet adhesion is one of the key indicators of blood compatibility with biomaterials.68 Platelet adhesion experiments were carried out using the platelet-rich plasma (PRP) method. A similar method was used in previous studies of platelet adhesion over surfaces of differing wettability.37,68 Figure 8 shows FESEM images of adherent platelets on hydrophilic (uncoated) (Figure 8a) and super-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b07831. XRD analysis of HMCCs and MCCs encapsulated with silver nanoparticles (Figure S1) and contact angle measurement of a water droplet a on stainless steel substrate (Figure S2) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address ‡

N.M.: Department of Mechanics and Wallenberg Wood Science Centre, KTH Royal Institute of Technology, Stockholm 11428, Sweden.

Author Contributions †

G.M. and N.M. contributed equally to this work.

Figure 8. Comparison of platelet adhesion on the uncoated SS surface and the SS surface coated with HMCCs. (a) FESEM image of the platelet adhesion on the SS surface (scale bar is 50 μm) with a magnified view in the inset (scale bar is 5 μm). It can be seen that the platelets have deformed and spread out over the surface. (b) FESEM image of the HMCC-coated SS surface (scale bar is 50 μm) with a magnified view in the inset (scale bar is 5 μm) after the platelet adhesion experiment. The surface is extremely clean, and no adhered platelets can be observed, except for one or two that can be seen at very few locations with the magnified views (inset).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the support by the Department of Science and Technology (DST), New Delhi, India. We thank the Thematic Unit of Excellence on Soft Nanofabrication at Indian Institute of Technology Kanpur for G


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providing us with the characterization facilities. We also acknowledge Rahul Bhardwaj from Advance Imaging and Rudra Kumar from IITK for assistance with the TEM images. Lars Wagberg is thanked for valuable inputs and discussions.

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