Bacteria Meet Graphene: Modulation of Graphene

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Bacteria meet Graphene: Modulation of Graphene Oxide Nano-sheets Interaction with Human Pathogens for an Effective Antimicrobial Therapy Valentina Palmieri, Francesca Bugli, Maria Carmela Lauriola, Margherita Cacaci, Riccardo Torelli, Gabriele Ciasca, Claudio Conti, Maurizio Sanguinetti, Massimiliano Papi, and Marco De Spirito ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00812 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017

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ACS Biomaterials Science & Engineering

Bacteria meet Graphene: Modulation of Graphene Oxide Nano-sheets Interaction with Human Pathogens for an Effective Antimicrobial Therapy Valentina Palmieria,c‡, Francesca Buglib‡, Maria Carmela Lauriolaa, Margherita Cacacib, Riccardo Torellib, Gabriele Ciascaa, Claudio Contic, Maurizio Sanguinettib, Massimiliano Papi a,c

*, Marco De Spirito a

a

Physics Institute, Catholic University of Sacred Hearth, Largo Francesco Vito 1, 00168 Rome (IT) b

Microbiology Institute, Catholic University of Sacred Hearth, Largo Francesco Vito 1, 00168 Rome (IT) c

Institute for Complex Systems, National Research Council (ISC-CNR), Via dei Taurini 19, 00185 Rome (IT) AUTHOR INFORMATION Corresponding Author *Corresponding author E-mail: [email protected] Prof. Massimiliano Papi, Physics Institute, Catholic University of Sacred Hearth, Rome, Italy Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

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KEYWORDS Graphene Oxide, DLVO theory, antibacterial, nano-blades, scaffold ABSTRACT The development of new pharmacological strategies that evade bacterial resistance became a compelling worldwide challenge. Graphene Oxide (GO) can represent the nanotechnology answer being economic, easy to produce and to degrade and having multi-target specificity against bacteria. Several groups tried to define the interaction between GO sheets and human pathogens. Unfortunately, controversial results from inhibition to bacterial growth enhancement have been reported. The main difference among all experiments evidences relies on the environmental conditions adopted to study the bacteria-GO interaction. Indeed GO, stable in deionized water, undergoes to a rapid and salt-specific DLVO-like aggregation that influences antimicrobial effects. Considering this phenomenon, interaction of bacteria with GO aggregates having different size, morphology and surface potential can create a complex scenario that explains the contrasting results reported so far. In this paper, we demonstrate that by modulating the GO stability in solution, the antibacterial or growth enhancement effect can be controlled on S. aureus and E. coli. GO at low concentration cut microorganisms membranes and, at high concentration, forms complexes with pathogens and inhibits or enhances bacteria growth in a surface potential-dependent manner. With the framework defined in this study, the clinical application of GO gets closer and controversial results in literature can be explained.

INTRODUCTION Antibiotic ineffectiveness represents a global health issue that demands the development of drugs circumventing microbial resistance mechanisms and attacking new bacterial targets1. Graphene Oxide (GO), a precursor of large-scale graphene synthesis, stable in water and easy to functionalize with drugs and proteins, attracted attention for its multi-targeting killing strategy,


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simple production and low cost2-5. Three possible antibacterial mechanisms have been observed: (i) GO sheets cutting bacteria membranes, (ii) inducing oxidative stress and/or (iii) wrapping and isolating bacteria from the external environment and nutrients6. However, from the first evidence of the antibacterial efficiency of GO in 20107-8, many papers followed but the overall efficacy of GO remained unclear, as described in recent reviews6, 9-10. Firstly, the effective concentration for growth inhibition is unknown, and size, dispersion state, surface functionality and type of microorganism change dramatically antimicrobial results6-7,

11-15

. Further, a series of studies

claimed no effect or even a growth enhancement when GO was added to bacteria growth medium16-22. As an example, considering effects on E. coli, it has been demonstrated that GO is antibacterial at concentrations below 10 µg/ml11, 23 or only above 100 µg/ml24-25 or that GO can enhance E. coli growth if administered at concentrations between 20 and 100 µg/ml17. This heterogeneity of results has been often explained by an inconsistent experimental design among different groups: in particular different methods used for GO synthesis leaving variable amount of contaminants in solution6. Though data for the residual substances in the GO material used in different studies are not always available, we can exclude this hypothesis since in a recent work Barbolina and colleagues demonstrated that contaminants do not influence the GO activity21. In this work, we point out the role of salt-dependent aggregation in the antibacterial efficiency of GO. Differently from antibiotics or macromolecules used for pharmacological applications, GO is a highly dispersible colloid in water but aggregates easily in solutions containing electrolytes. Wu and colleagues found that sodium, calcium and magnesium ions cause aggregation of GO at a critical concentration of coagulation of 188 mM, 2.6 mM and 3.9 mM respectively and that divalent cations bridge functional groups at the edges of GO26. These aggregates have macroporosity and shape that can change the pharmacological efficacy of GO. In order to perform


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antimicrobial tests, bacteria are suspended either in water, NaCl, PBS or in physiologically relevant cations; all these different experimental conditions drastically alter GO stability and might cause the heterogeneity of the results reported in literature27. Even if the colloidal physics and the “Plate−Plate DLVO interaction” describing the GO sheets aggregation in electrolyte solutions has been studied26, 28, the role played by GO stability has not been fully considered in the outcomes of microbiological studies. To assess the role of aggregation and material surface potential in bacteria-GO interaction, here we test GO against the Gram positive S. aureus and the Gram negative E. coli. We expose these two species to concentrations of GO ranging from 3 to 200 g/ml, in two different incubation conditions: in solutions without nutrients (ultrapure water, PBS, NaCl, MgCl2 and CaCl2) or directly in growth medium (LB broth). We analyze the growth of microorganisms, and characterize GO effects on cells with Atomic Force Microscopy and Colony Forming Units Assay and show that GO effects on bacterial growth depend on its stability. At concentrations below 6 g/ml, GO is effective in all solutions examined, and acts as a knife that cuts bacterial membranes. At low concentration, GO is indeed stable and few ions in solution favor collisions between bacteria and GO edges. As GO concentration increases, the GO killing efficacy increases only in water; in other solutions GO does not impact microbial growth because of aggregates that shield GO edges. However, when GO cluster size becomes much larger than bacteria diameter, GO aggregates wrap bacteria and impede their growth. This occurs above 100 g/ml in NaCl, CaCl2 and MgCl2; at this concentration, high surface energy causes bacteria trapping. In PBS, GO displays a slight variation of surface charge and bacteria are still adsorbed on GO clusters. In this condition, as demonstrated by CFU counting, the bacterial growth is fostered. Further we demonstrate that also in presence of nutrients the instability of GO is a


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crucial parameter influencing its activity. In summary, we demonstrate the way GO effects can be modulated by the surrounding solution, in order to facilitate GO application in clinics and in environmental science. MATERIALS AND METHODS Dynamic Light Scattering and Zeta potential measurements GO solutions were prepared as reported elsewhere29 at 2 mg/ml (Graphenea) and sonicated for 30 minutes with a probe-sonicator at 50 W (VC50 Sonics and Materials UK) to reduce sample polydispersity. Solutions were characterized with Dynamic Light Scattering with Zetasizer Nano ZS (Malvern, Herrenberg, Germany) equipped with a 633nm He–Ne laser and operating at an angle of 173°. Solvent-resistant micro cuvettes (ZEN0040, Malvern, Herrenberg, Germany) have been used for experiments with a sample volume of 40 l. The measurements were performed at a fixed position (4.65 mm) with an automatic attenuator and at a controlled temperature (20°C) as reported previously30. For each sample, five measurements were averaged, the diffusion coefficient D has been retrieved through cumulants analysis from autocorrelation functions31. The equivalent Hydrodynamic Radius (Z- Average size) was obtained by the Stokes-Einstein equation31. Data analysis was performed by Malvern Zetasizer software. The -potential was calculated from the electrophoretic mobility by means of the Henry correction to Smoluchowski's equation with Data analysis was performed by Malvern Zetasizer software.Zetasizer Nano ZS (Malvern, Herrenberg, Germany) averaging 5 measurements29.


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Cell viability tests E. coli (ATCC strain 25922) and S. aureus (ATCC strain 29213) have been used to perform viability tests. Bacteria were grown in LB medium at 37 °C overnight and cells were harvested via centrifugation (4000 rpm for 10 min). Cells were washed three times with deionized water to remove residual macromolecules and other growth medium constituents. The pellets were then suspended in ultrapure water, NaCl Solution (0,9%), D-PBS buffer, CaCl2 (10 mM) and MgCl2 (10 mM), Luria Bertani (LB) Broth solutions. Hereafter we will refer to these buffers by using ddH2O, NaCl, PBS, CaCl2, MgCl2, LB. Bacterial cell suspensions were diluted in the specific buffers or solutions to obtain cell samples having a Mc Farland turbidity of 0,7 corresponding to 107 −108 CFU/mL. 180 l of cells in different buffers were incubated with 20 l of fresh GO suspensions at room temperature for 4 h without shaking, or for 24 h in LB broth. Growth in LB broth has been followed by measuring OD with Cytation 3 Cell Imaging Multi-Mode Reader. The viability was evaluated by the colony counting method. Briefly, series of 10-fold cell dilutions (100 μl each) were spread onto LB plates, and let grow overnight (12 hours) at 37 °C. Colonies were counted, and compared to those on control plates to calculate changes in the cell growth inhibition. Atomic Force Microscopy Samples were prepared as explained elsewhere32. Briefly, 20 l of samples were deposited on sterile cover glass slides, air dried and washed once with ultrapure water (37°C) to remove salt deposits interfering with the measurements. After sample preparation, measures were immediately performed in ddH2O with a NanoWizard II atomic force microscope (JPK Instruments AG, Berlin, Germany). The images were acquired using silicon cantilevers with high


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aspect-ratio conical silicon tips (CSC37 Mikro-Masch, Tallinn, Estonia) characterized by an end radius of about 10 nm and a half conical angle of 20°. Cantilevers with a nominal spring constant of about 𝑘 = 0.4 N/m, were accurately calibrated as previously reported32. A large scan area (20 × 20 𝜇m) was imaged in order to determine the number of bacteria on aggregates while for bacteria integrity areas of 20 × 20 𝜇m were imaged. To characterize the surface properties of aggregates the Root Mean Square Roughness (RMS) was calculated as reported before 32. RESULTS Effect of environment surroundings on GO stability To test the activity of GO in different environmental conditions we exposed bacteria to GO in different solutions (ultrapure water, PBS, NaCl, MgCl2 and CaCl2).

In Figure 1 the GO

characterization by means of DLS and AFM is presented. In Figure 1A-E images of cuvettes containing GO in different solutions (200 g/ml) are reported immediately and after 24 hours after dilution from the GO stock solution in ddH2O. In ultrapure water, GO is well dispersed in a single layered state with an average thickness of 0.8 nm and an average surface area of 0.19 m2 measured from AFM images (Figure 1A). In the cuvette, the solution appears homogeneous with a light brown color and remains stable after 24 hours (Figure 1A). GO displays stable hydrodynamic radius in ddH2O between 3 and 200 g/ml measured from DLS (Figure 1F and Supplementary Figure S1), and a  of -37.8 mV (Figure 1G).


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Figure 1. Stability of GO in different solutions (A-E) Pictures of cuvettes containing GO in different solutions (200 g/ml) immediately after resuspension or after 24 hours and corresponding AFM height images (in liquid) of GO in ddH2O (A), PBS (B), NaCl (C), MgCl2 (D) and CaCl2 (E). Hydrodynamic radius of GO in different solutions obtained with DLS (F). Zeta potential of GO in different solutions compared to Z potential of S. aureus and E. coli bacteria in the same solutions (G). Thickness and RMS of GO aggregates obtained from AFM images (H and I, respectively). Aggregates stability in different solutions after 0-10-30 seconds of vortexing (L).

In solutions containing electrolytes, GO quickly aggregates, as it is visible from photographs of cuvettes: aggregates form immediately after the dilution of GO in PBS, NaCl, CaCl2 and MgCl2 solutions and precipitate with time (Figure 1B-E) after 24 hours. GO aggregates have different properties in respect to GO sheets and their surface area, roughness and zeta potential will influence the effect on bacteria6. The size and surface properties of aggregates varies with the kind of ions in solution. Aggregates form in a concentration dependent manner (Figure S1). After


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24 hours, a stable size of GO has been measured below 6 g/ml, with a size always below 750 nm. Above 12 g/ml, divalent cations cause systematically the formation of larger aggregates. For all solutions considered, there is an abrupt increase of size over 50 g/ml (Figure S1). At the highest concentration tested, 200 g/ml, largest aggregates are formed in presence of CaCl2, followed by aggregates in MgCl2, PBS and NaCl. Measured aggregates thickness from AFM images (Figure 1A-E) confirm that largest aggregates are formed in CaCl2 with an average thickness of 81 nm, followed by MgCl2 (58 nm), PBS (51 nm) and NaCl (49 nm) at 200 g/ml. It should be noted that aggregates formed in CaCl2 are much thicker in respect to other buffers. Wu and colleagues have extensively studied this mechanism and demonstrated how in presence of calcium divalent cations, the process of aggregation differs because of a calcium-specific crosslinking of functional groups on GO26. Aggregates display a different stability in solution as shown in Figure 1L. Indeed, PBS aggregates reduce size of 50% after vortexing for 10 seconds, NaCl aggregates reduce size of 40% after vortexing for 30 seconds while CaCl2 and MgCl2 aggregates remain insoluble and are the most stable (Figure 1L). The aggregation of GO occurs because of a decrease of repulsive forces between GO sheets, due to the cations in solution: cations shield surface negative charges as is visible from  values in Figure 1G. This screen effect occurs mostly in solutions of divalent cations were  is –9,9 mV for MgCl2 and – 8,0 mV for CaCl2 and is less visible in PBS (- 25,8 mV) and NaCl (-19,4 mV).  of bacteria is reported in Fig 1G. In general, for most bacteria, the net surface charge is negative, we measured  around 20 mV for both S. aureus and E. coli in water that decreases when increasing ions in solution. Since  can decrease also because of cell death, we performed OD growth curves of bacteria after incubation in different buffers (data not shown) and confirmed that bacteria are still able to grow and duplicate in each solution considered. Therefore, the observed decrease of  is, as in


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the case of GO, due to the positive charges attracted by the negative cell membrane. Since bacterial surface attachment follows surface topography, RMS of aggregates has been calculated from AFM images (Figure 1A-E). Values of RMS are shown in the graph in Figure 1I. The RMS goes from values below 0.1 nm in water to 13.1 nm in PBS and NaCl, 26.5 nm in MgCl2 and 42.5 nm in CaCl2 at 200 g/ml. The surface features are crucial factors affecting bacterial colonization since increased surface area and deep terrains result in greater adhesion33.

Figure 2. Normalized CFU assay results and AFM images of E. coli exposed to GO in ddH2O (A), PBS (B) and NaCl (C) solutions. Sketches on graphs highlight the different effects of GO.

Antimicrobial effects depend on concentration and solution considered GO effects on microorganisms vitality have been analyzed for two model organisms: gramnegative E. coli and gram-positive S. aureus. Firstly, the activity of GO in ddH2O, PBS and NaCl has been compared (Figure 2). These solutions have been indifferently used for bacterial washing and resuspension procedures during vitality tests though GO has different stability7, 11, 13, 24. As is visible in Figure 2, independently


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from the solution used, there is a clear growth reduction yet at GO concentration of 3 g/ml. The effect is clearer in PBS and NaCl in respect to ddH2O. From AFM images, we see disruption of the cellular integrity and debris in PBS, NaCl and partially also in ddH2O (Figure 2 for E. coli and Figure S2 for S. aureus results). If we increase the GO concentration, three different trends are visible. In ddH2O, there is a decrease of bacteria vitality with increase of GO concentration. Cell vitality is 60% for S. aureus and 90% for E. coli at 3 g/ml of GO and gradually reaches total loss of growth for S. aureus and a consistent inhibition for E. coli (50%) over 100 g/ml. From AFM images cells appear disrupted after treatment with GO in ddH2O. In PBS, the GO sheets still cut the membranes of bacteria at concentrations below 12 g/ml (Figure 2B for E. coli and Figure S2 for S. aureus results). Vitality at concentrations below 12 g/ml is around 20% for both S. aureus and E. coli. Increase of the GO concentration causes a progressive loss of antibacterial effect. Moreover, a progressive growth enhancement over 25 g/ml for S. aureus and over 100 g/ml for E. coli is visible. Finally, in NaCl the GO induces a loss of 80% live cells for both S. aureus and E. coli at concentrations below 12 g/ml. Then the antibacterial activity is gradually lost between 12 and 50 g/ml and restored over 100 g/ml of GO (Figure 2B) where inhibition is around 75% for S. aureus and 45% for E. coli. From AFM images, it is visible that in PBS and NaCl, bacteria structure is lost at concentration below 12 g/ml, where cellular debris are visible. On the other hand, as concentration of GO increases, bacteria appear bound to GO aggregates and their cell integrity is not affected (AFM Panel in Figure 2 for E.coli and Figure S2 for S. aureus). Over the bacteria surface wrinkles are visible, indicating GO sheets covering cell membranes (Figure 5 C-D).


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Figure 3. Normalized CFU assay results and AFM images of E. coli exposed to GO in MgCl2 (A) and CaCl2 (B) solutions.

Modulation of GO efficacy: divalent cations favor GO-bacteria interaction To clearly demonstrate how the antibacterial effect is influenced by the charge and stability of aggregates in solution we quantified GO effects on bacteria in presence of 10mM MgCl 2 and CaCl2 (Figure 3) that immediately cause the irreversible aggregation of GO. In these solutions, different phenomena are visible with GO great efficacy at extremely low (100 g/ml) concentration. The interaction between GO and bacteria is favored and the bactericidal effect is greatly enhanced in respect to other solutions. From AFM images, we see again a disruption of bacteria integrity at low concentrations and an attachment of bacteria to scaffolds at higher concentrations (Figure 3 for E. coli and Figure S2 for S. aureus). Bacteria are distributed over the entire surface of the flakes. From optical microscopy measurement bacteria appear so tightly bound to aggregates that there are no bacteria visible in the field of view other than those on aggregates (data not shown). On the surface of bacteria are mostly as individual


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cells or small colonies. The GO in presence of calcium ions cause an enhanced effect in respect of magnesium, even at intermediate concentrations. Indeed calcium is known to promote both specific and non-specific interactions with protein and polysaccharide adhesion molecules at the bacteria surface 34. GO efficacy is limited in presence of growing bacteria Two different protocols have been adopted in literature to test the effects of GO: exposition of bacteria to GO in solutions without nutrients (like in ultrapure water, PBS or NaCl) or incubation of bacteria with GO directly dissolved in growth medium (Figure S3A). As we demonstrated, the first procedure allows to define the GO effects in a “static” environment, and to characterize the interaction between bacteria and the nanomaterial. Conversely, antibiotics are conventionally tested in conditions in which growth is fostered to simulate in vivo conditions during infections. Data in literature, resumed in Fig. S3B, indicate generally a lack of effect of GO in broth, therefore we investigated GO antibacterial properties and related to its stability in LB solution. Soon after dissolution in LB, the GO quickly aggregates as is visible from pictures of cuvettes in Figure 4A. Aggregates create a deposit after 24 hours and cannot be suspended with vortexing. We measured size of aggregates with DLS and observed a stable hydrodynamic radius of 1.5 m at concentrations below 100 g/ml, which increases over 3 m at 200 g/ml. The ZP of aggregates is -14.753.74 mV, indicating that negative GO surface charges are screened by broth constituents that cover basal planes of GO, as previously reported35. Representative height AFM image of LB aggregates at 200 g/ml is reported in Fig. 4C, the average thickness is 173 nm15 nm and RMS 65.8 nm2.3nm. Therefore, aggregates in LB broth are larger in respect to those formed in other solutions (Figure 1).


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We

Figure 4. (A) Pictures of cuvettes of GO in LB at different concentrations. (B) Hydrodynamic radius measured with DLS. (C) AFM height image of GO aggregate in LB (200 g/ml), 40x40 m.(D) Growth curves of S. aureus and E. coli incubated with different GO concentrations in LB, OD has been normalized to subtract the aggregation of GO in LB. AFM deflection image of GO rafts (25g/ml) after 5 hours of incubation (40x40 m in (E) and 10x10m in (F)) or after 24 hours of incubation (G).

have analyzed the effects of GO in LB against E. coli and S. aureus and we report in Figure 4D the growth curves in presence of different GO concentrations. As is visible the antimicrobial effect of GO is concentration dependent after few hours of incubation but is not visible after 24 hours of incubation, when all concentrations considered reach the control OD. We analyzed morphology of E. coli and S. aureus cells with AFM and observed GO rafts trapping cells after 5 hours (Figure 4E), as expected from the Zeta Potential value. In Figure 4F, yellow arrows


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highlight the GO sheets below E. coli cells. In Figure 4G is visible how GO sheets are completely covered by bacteria after 24 hours (Figure 4G). Therefore, in this “dynamic” situation the GO aggregates limit growth and trap bacteria initially, but this effect is exhausted when the number of bacteria saturate the available GO surface, explaining the poor effects observed in literature when this protocol has been used. DISCUSSION The use of GO as antibiotic material against bacteria has recently attracted wide research interest 36-37

. Our data show that buffer solution can markedly influence vitality test and that a

coexistence of three different GO effects occurs. Furthermore, we demonstrate how results are influenced by the protocol used. We considered two different protocols, based on literature data, schematized in Figure S3A. In the first protocol, the bacteria have been exposed to GO in solutions in which growth is not promoted. We will refer to this as “static” incubation. Oppositely, microorganism treated with GO in LB broth, are actively growing thanks to the nutrients (“dynamic” incubation). In the static incubation, the greatest bactericidal effect of GO is visible below 6 µg/ml. At these concentrations, GO acts as a cutter of bacteria membrane in all the experimental conditions studied as shown in AFM images (S. aureus in Figure 5 A and E. coli in Figure 5 B). The GO mediated extraction of membrane lipids has been previously demonstrated with both molecular dynamics simulations38 and in vitro studies that reported the mechanical disruption of cell membranes, the leakage of intracellular material and eventually cell death. It should be pointed out that in our study, the size of GO sheets is smaller than bacteria, therefore the blade effect is the predominant mechanism. In water solution, the antibiotic effect is mild since GO and bacteria have a comparable low  and repulsive forces act between them.


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Figure 5. Representative AFM error images of S. aureus (A-C) and E. coli (B-D) after GO treatment. At low GO concentration membrane cut causes leakage of intracellular material and loss of bacteria integrity (A-B). At high GO concentration in presence of salts bacteria are wrapped by GO sheets aggregates (C-D). Scale bar is 1 µm.

The greatest effect in reduction of bacteria vitality at low concentrations is visible in the presence of divalent cations. This is mainly a charge-mediated effect. Castrillon and colleagues studied the interaction between GO and E. coli cellular membranes using atomic force microscope (AFM) and demonstrated that interactions are predominantly repulsive and this repulsion occurs between deprotonated carboxylic acid groups in GO and negative cell membrane of bacteria39. The increase of Z potential due to cations on GO surface reduces repulsion and favors collisions between sheets and bacteria causing an enhanced antibacterial efficiency, independently of the kind of bacteria investigated. In water, the effect varies between the two microorganisms since


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bacteria have different organization of bacteria cell wall: Gram negative bacteria are less sensible, since possess a double layer of membranes which act as a further protective shield against GO sharp edges40. Overall this very low concentration needed for GO antimicrobial activity points out the great potential of GO for the development of antibiotic therapies with limited side effects. Increase of the GO concentration causes different outcomes. In water solution, GO is highly stable and the higher the concentration, the higher the number of edges cutting cell membranes, the higher the vitality loss. In presence of salts, GO forms large clusters in a concentration- and ions-dependent fashion and antibacterial effects will depend on GO concentration, charge, dispersibility and aggregative state 6. When bacteria face aggregates rather than single GO planes, cutting edges are screened inside these aggregates and exposed edges are formed by multiple layers of GO sheets. In this case the energy barrier to pierce membranes is greater than single graphene sheets and the blade effect is progressively cancelled 6. This observation is supported by in vivo experiments of graphene oral administration that reported that 10 and 100 μg/d of graphene exposures caused lower changes in gut microbial community compared to 1μg/d41. In this paper, authors hypothesized an effect due to aggregative state of graphene. However large GO aggregates can bind bacteria and inhibit their growth with a second mechanism of action of GO, the so-called wrapping strategy (S. aureus in Figure 5 C and E. coli Figure 5 D). In this phenomenon bacteria are insulated by a GO “blanket” from external environment, transport through cell membrane, movements and proliferation are impeded but, as shown by AFM images, bacteria retain their architecture and remain attached to the surface 11, 42. Since divalent cations influences bacterial adhesion, calcium and magnesium cations cause the most evident trapping effect in respect to saline solution. The wrapping is most efficient for Gram Positive S. aureus maybe due to specific cell wall architecture. Indeed, as the constituents


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of bacteria cell wall is complex, and the extent to which a cell surface molecule participates in adhesion to a substratum depends on the species considered and the variety of results in literature on different microorganisms can be explained by this phenomenon 34. Calcium cations are more effective in wrapping than magnesium. This can be explained both by an increased size of aggregates and a specific role in adhesion process. Indeed calcium is involved in specific adhesive interactions that cannot be replaced by other cations like regulation of adhesion proteins in a wide range of bacteria34. Once that the attachment is tight, bacteria cannot move or proliferate. In summary, at concentrations >100 µg/ml, GO aggregates formed in presence of divalent cations have the ability of wrapping bacteria, impeding their growth without releasing bacterial debris in the environment (Figure 6). This can represent an excellent strategy for water remediation purposes. In a recent review it is pointed out how GO represents an excellent carbon material for its better dispersion in water that allows increased available surface to remove chemical and microbiological9. Following these data, the amount of cations in solution can be exploited to determine the degree of bacteria trapping and also to maximize electrostatic interaction and strength of binding. Moreover, for this specific application the blade effect reduction is desirable since no bacteria constituents are released in water but bacteria remain totally trapped in a GO “filter”. In principle by modulating also the aggregates roughness other than surface charge one can be selective against determined bacteria. Indeed the RMS of the aggregates determines the degree of deep terrains where bacteria can be trapped33. Finally, we discovered that if the bacteria and GO are weakly interacting, GO can be used a scaffold to foster bacteria growth. In PBS, the shielding of edges inside aggregates is combined to an inefficient wrapping caused by the poor stability of the GO scaffold in PBS (see Figure S1) and the low strength of the bacteria-GO binding. In this buffer, the link between the bacteria and the GO is


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not tight and GO aggregates become a scaffold for attachment and growth of cells not stuck by GO interaction. This effect occurs also at intermediate concentration of NaCl and can be exploited for probiotic therapies. For example, Chen and colleagues17 reported an enhancement of bacteria growth, that is selective toward gut B. adolescentis while having an antagonistic effect on E. coli and S. aureus. They performed experiments in NaCl solution and interestingly we have also seen a slight enhancement of growth at concentration comprised between 25 and 50 g/ml in saline solution. In the dynamic protocol, i.e. when growth is promoted, the GO faces an increasing number of growing bacteria. We resumed data available from literature on E. coli in Figure S3B, and related concentration, efficacy and solution used for incubation. As is visible, GO is more effective in ultrapure water and is less effective in saline

11, 43

. Oppositely, data

obtained after direct administration of GO in LB broth indicate a lack of antimicrobial activity or even a growth enhancement (negative efficacy in Figure S3B)

16-17, 35

. We observed a great GO

instability in LB broth and measured a hydrodynamic radius of aggregates larger than any other solutions. Growth experiments and AFM imaging indicate that in LB the GO acts as bacteria trapper but that once the surface availability is saturated by the growing bacteria, the effect is cancelled (Figure 5D). In LB broth, we obtained only a “temporal delay” of the bacterial growth. Such delay increases with the GO concentration since initially the bacteria are well trapped by the GO aggregates. However, the bacteria that do not interact with the GO are still able to reproduce by cell division in the broth and, as soon as the GO cluster surfaces are completely covered, the new bacteria can freely grow and reach the same final cell number of the untreated sample. Interestingly this phenomena is in agreement with the observations reported in

15

that

reported a E. coli inhibition in MH broth only after 5 hours and a complete cell number recovery after 24 hours.


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CONCLUSIONS


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In recent years, significant research related to antibacterial properties and effects on eukaryotic cells of GO and GO-based materials has been conducted

44

. Despite substantial efforts, results

are controversial and a framework clarifying all effects reported in literature against bacteria remains to be defined6. Our data indicate that any buffer solution utilized during the GO-bacteria interaction alters specifically the GO surface zeta potential and the consequent GO clusters size and structure. This implies that during the incubation procedures microorganisms interact with an unstable nanomaterial shaped by the physicochemical conditions. We performed our experiments reproducing the most frequently reported environmental conditions and GO concentrations and we found effects drastically different since GO can act both as antibiotic and growth promoter, giving the surprising possibility to finely modulate the GO effects on bacteria and to produce versatile applications in the environmental and medical sciences (as shown in Figure 6). At low GO concentration, as long as the GO flakes are stable, the presence of


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counterions promotes bacteria-GO collisions (Figure 6A). In this situation, the low toxicity makes GO an ideal nanomaterial to develop antibiotic formulations. Differently at higher GO concentrations, counterions produce large GO clusters that can produce two opposite effects. At low , GO clustering forms floating scaffolds able to enhance bacterial growth while, at high , GO forms scaffolds able to inhibit the bacterial growth. Further studies should define the pharmacokinetics in biological fluids, and other effects of experimental surrounding such as presence of nutrients or incubation with agitation in the efficacy of GO. The GO versatility can be exploited as a promising strategy for the development of treatments against multidrug resistant bacteria, water remediation and probiotic therapies.

Figure 6. Scheme of GO interaction with bacteria (A) At concentrations below 6 g/ml, GO is well dispersed in all the solutions investigated and acts as a cutter of bacteria membrane. The effect is similar for E. coli and S. aureus in all solution except for ddH2O, where GO has higher antibacterial efficacy for S. aureus. (B) The bacteria adsorb on GO scaffolds at concentration above 100 g/ml in a dependent manner. The highest adsorption occurs at higher . Yellow arrows indicate GO cluster rims. Counts of bacteria per aggregate area have been obtained from AFM images using ImageJ software.


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ACKNOWLEDGMENT Experiments have been performed at the LABCEMI Microscopy Facility (UCSC, Rome, Italy). We are extremely thankful to Mario Amici for the technical support during experiments. This research has been founded by ERC-POC Vanguard grant 664782. ASSOCIATED CONTENT Supporting

Information.

The

following

files

are

available

free

of

charge.

Figure S1 GO size in different solutions at concentrations; Figure S2 AFM images of S. aureus exposed to GO in ddH2O, PBS, NaCl, MgCl2 and CaCl2 solutions. Figure S3 A) Schematic representation of protocols adopted for bacteria exposure of GO B) Effects of GO administered in different concentration and solutions to E. coli. Data have been obtained from literature. ABBREVIATIONS AFM Atomic Force Microscopy; CaCl2 Calcium Chloride; CFU Colony Forming Unit; ddH2O double-distilled water; DLS Dynamic Light Scattering; DLVO Derjaguin-Landau- VerweyOverbeek; GO Graphene Oxide; LB Luria-Bertani; MgCl2 Magnesium Chloride; NaCl Sodium Chloride; OD Optical Density; PBS Phosphate Buffered Saline; RMS Root Mean Square

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For Table of Contents Use Only Bacteria meet Graphene: Modulation of Graphene Oxide Nano-sheets Interaction with Human Pathogens for an Effective Antimicrobial Therapy Valentina Palmieria,c‡, Francesca Buglib‡, Maria Carmela Lauriolaa, Margherita Cacacib, Riccardo Torellib, Gabriele Ciascaa, Claudio Contic, Maurizio Sanguinettib, Massimiliano Papi a,c

*, Marco De Spirito a

TABLE OF CONTENTS GRAPHIC Graphene Oxide (GO) effects on bacteria are modulated by concentration, surface zeta potential (ZP) of the material and growth conditions of bacteria. When bacteria are not growing, at low concentration GO is a powerful antibiotic, while at high concentration by changing surface charge GO becomes a scaffold which favours bacteria growth (SP-10 mV). In presence of nutrients, the GO also traps bacteria, but its action is limited in time and is saturated by high number of bacteria.


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Bacteria Meet Graphene: Modulation of Graphene

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