# Historical perspective Probing the threshold of membrane damage and cytotoxicity effects induced by silica nanoparticles in Escherichia coli bacteria

Marion Mathelié-Guinlet, Laure Béven, Fabien Moroté, Daniel Moynet,

Christine Grauby-Heywang, Ibtissem Gammoudi, Marie-Hélène Delville,

Touria Cohen-Bouhacina

► To cite this version:

Marion Mathelié-Guinlet, Laure Béven, Fabien Moroté, Daniel Moynet, Christine Grauby-Heywang, et al.. Historical perspective Probing the threshold of membrane damage and cytotoxicity effects induced by silica nanoparticles in Escherichia coli bacteria. Advances in Colloid and Interface Science, 2017, 245, pp.81-91. 10.1016/j.cis.2017.04.012. hal-01540158

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Submitted on 19 Jun 2017

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# Probing the threshold of membrane damage and cytotoxicity effects induced by silica nanoparticles in *Escherichia coli* bacteria

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Marion Mathelié-Guinlet<sup>a,b</sup>, Laure Béven<sup>c</sup>, Fabien Moroté<sup>a</sup>, Daniel Moynet<sup>d</sup>, Christine Grauby-Heywang<sup>a</sup>, Ibtissem Gammoudi<sup>a,e</sup>, Marie-Hélène Delville<sup>b,*</sup>, Touria Cohen-Bouhacina<sup>a,c,**</sup>

## ABSTRACT

Keywords:

E. coli

Silica nanoparticles

Membrane

Toxicity

AFM

The engineering of nanomaterials, because of their specific properties, is increasingly being developed for commercial purposes over the past decades, to enhance diagnosis, cosmetics properties as well as sensing efficiency. However, the understanding of their fate and thus their interactions at the cellular level with bio-organisms remains elusive. Here, we investigate the size- and charge-dependence of the damages induced by silica nanoparticles (SiO₂-NPs) on Gram-negative *Escherichia coli* bacteria. We show and quantify the existence of a NPs size threshold discriminating toxic and inert SiO₂-NPs with a critical particle diameter (Φ<sub>c</sub>) in the range 50nm–80nm. This particular threshold is identified at both the micrometer scale *via* viability tests through Colony Forming Units (CFU) counting, and the nanometer scale *via* atomic force microscopy (AFM). At this nanometer scale, AFM emphasizes the interaction between the cell membrane and SiO₂-NPs from both topographic and mechanical points of view. For SiO₂-NPs with Φ > Φ<sub>c</sub> no change in *E. coli* morphology nor its outer membrane (OM) organization is observed unless the NPs are positively charged in which case reorganization and disruption of the OM are detected. Conversely, when Φ < Φ<sub>c</sub>, *E. coli* exhibit unusual spherical shapes, partial collapse, even lysis, and OM reorganization.

## 1. Introduction

For the past decades, nanotechnologies field has experienced a fast and growing development in many areas (microelectronics, medical imaging, cosmetics...) because of the specific physico chemical proper ties of nanoparticles (NPs). Though, this enthusiasm began to be questioned as NPs might put environmental safety and human health at risk by interacting with biological systems and affecting their behavior at the cellular level [1 7]. The diversity of NPs in terms of composition, shape and size/diameter (Φ) challenges politicians for regulating their safe use, and scientists for understanding how they interact with living organisms [8,9]. Such regulation and understanding are, though, necessary for the safe development of various applications like diagnosis in medicine [10 12], smart textiles conception [13,14], environmental treatments and detection sensors [15,16].

Among widely used biological systems, *Escherichia coli* is the most

thoroughly studied species of bacteria. Ubiquitous in the environment, *E. coli* are Gram negative bacteria which, along with the cytoplasmic membrane and a peptidoglycan layer, possess an outer membrane (OM) composed of a phospholipid bilayer, containing lipopolysaccharide (LPS) molecules and proteins [17]. Potential human infections and diseases need to be considered as a consequence of exposure to several *E. coli* strains. The strain *E. coli* MRE 162 that we used in this work was first studied during the Lyme Bay trials in 1966 in order to mimic real biological attacks and to determine how United Kingdom (UK) popula tions could be at risk. Releasing massive amount of such bacteria, UK militaries reported chest and blood infections, caused by inhalation of *E. coli* MRE 162, especially in individuals highly susceptible to disease and exposed to contaminated clouds [18].

Studies on the antibacterial activity of NPs towards other *E. coli* strains showed that diverse metal oxides [19], iron [20] copper and silver [21] based nanomaterials exhibit a bactericidal activity. Major

<footnote><sup>a</sup> Univ. Bordeaux, CNRS, LOMA, UMR 5798, 351 cours de la Libération, 33400 Talence, France</footnote>

<footnote><sup>b</sup> Univ. Bordeaux, CNRS, ICMCB, UPR 9048, 87 avenue du Dr Albert Schweitzer, 33608 Pessac, France</footnote>

<footnote><sup>c</sup> Univ. Bordeaux, INRA, UMR 1332 Biologie du Fruit et Pathologie, 33140 Villenave-d'Ornon, France</footnote>

<footnote><sup>d</sup> Univ. Bordeaux, INSERM U1035, 146 rue Léo Saignat, 33000 Bordeaux, France</footnote>

<footnote><sup>e</sup> Cellule de transfert NanoPhyNov, ADERA, LOMA, 351 cours de la Libération, 33400 Talence, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Correspondence to: T. Cohen-Bouhacina, Univ. Bordeaux, CNRS, LOMA, UMR 5798, 351 cours de la Libération, 33400 Talence, France.
E-mail addresses: marie-helene.delville@icmbc.cnrs.fr (M.-H. Delville), touria.cohen-bouhacina@u-bordeaux.fr (T. Cohen-Bouhacina).</footnote>
toxicity mechanisms include the disruption of the bacterial membrane integrity leading to the leakage of intracellular components [22], the production of reactive oxygen species (ROS) damaging bacterial constituents [21] and/or the dissolution of NPs into their constituting ions interfering with the bacterial metabolism [23]. These mechanisms depend on multiple factors (composition, chemical functionalities, shape), especially the size and charge of NPs [29, 33]. For instance, Beddoes et al. have presented an overview of in vivo and in vitro studies on human cells, and membrane models simulations, reporting that (i) small NPs (tens of nm) translocate very efficiently through the membrane causing cellular damage while large NPs (hundreds of nm) exhibit strong cellular uptake without toxicity; (ii) positively charged NPs disrupt membrane integrity and are more toxic than negatively charged NPs [8].

Because of their relatively low toxicity towards eukaryotic cells, their biocompatibility and their easy surface modifications, silica NPs (SiO₂ NPs) are good candidates for biomedical imaging, drug delivery, and biosensor applications [34,35]. Interestingly, SiO₂ NPs, not per se but functionalized with either photosensitizing molecules or antibiotics, or anchored to nanohybrid materials are promising both in bacterial detection [36] and antibacterial activity [37]. Despite this great potential, the interactions of non functionalized SiO₂ NPs with bacteria are not well documented in the literature. Most studies on toxicity of such NPs towards microorganisms rely on colorimetric methods or on Colony Forming Units (CFU) counting which allow the quantification of the minimum bactericidal concentration and the determination of the percentage of dead cells [38,39]. However, even if the death of cells is a key parameter, their behavior in the presence of bactericidal com pounds is also crucial to investigate and to understand the underlying toxicity mechanisms and to distinguish between the effects of different parameters such as NPs size and charge.

Some authors used transmission electron microscopy (TEM) to observe the morphological damages induced to *E. coli* cells by ZnO and Ag NPs [40,41]. Confocal Raman spectroscopy molecular inter pretation for such damages was also performed for GaN NPs interacting with different bacterial strains [22]. In this context, atomic force microscopy (AFM) is also another technique which provides comple mentary opportunities (i) to observe biological systems at a nanometer scale, both in air and in physiological environment, and (ii) to probe their nanomechanical properties in real time [42, 44]. Under these conditions, AFM is thus a pertinent technique to analyze bacteria morphological and rheological damages induced by potentially anti bacterial agents, such as NPs, with details at the nanoscale.

In the present work, our main objectives were to better understand the process of interaction of SiO₂ NPs with *E. coli* MRE 162, for which, to our knowledge, it is the first study of exposure to NPs. We aimed at determining a potential size and charge threshold, for which SiO₂ NPs would be toxic towards *E. coli*. NPs of 100 nm in diameter and larger have been shown to exhibit similar behaviors so that the herein considered range (4 nm to 100 nm) will focus on all the opened questions about size dependent biotoxicity of NPs [45,46]. These SiO₂ NPs were characterized in terms of size distribution and aggrega tion state in solution. We first quantified the bacterial viability upon incubation with such particles. Then, we focused on how the presence of SiO₂ NPs of different sizes and charges disturbs the bacteria morphology and the mechanical properties of their membrane, by taking advantage of AFM in tapping mode and finally correlated these data with the viability results.

## 2. Results

### 2.1. Size and charge characterization of SiO₂ NPs

Dynamic Light Scattering (DLS) measurements (Table 1) revealed that, for the largest SiO₂ NPs⁻ (Φ ≥ 50 nm), the average hydrody namic diameter was larger than the primary particle sizes obtained by

<table><caption>**Table 1**

Characterization of SiO₂-NPs, both commercial and home-made, by DLS and zeta potential measurements and statistics on TEM pictures.</caption><thead><tr><th rowspan="2">Name</th><th colspan="3">Diameter (nm)</th><th rowspan="2">Zeta potential (mV)</th></tr><tr><th>Manufacturer</th><th>TEM</th><th>DLS</th></tr></thead><tbody><tr><td>SiO₂-NPs</td><td>4 (large distribution)</td><td>aggregated</td><td>3</td><td rowspan="5">53.3</td></tr><tr><td>SiO₂-NPs</td><td>10 ± 5</td><td>12 ± 4</td><td>7</td></tr><tr><td>SiO₂-NPs</td><td>50 ± 10</td><td>53 ± 12</td><td>68</td></tr><tr><td>SiO₂-NPs</td><td>80</td><td>81 ± 4</td><td>90</td></tr><tr><td>SiO₂-NPs<br>100</td><td>100 ± 30</td><td>113 ± 11</td><td>124</td></tr><tr><td>SiO₂-NPs<sup>+</sup><br>100</td><td>Home made</td><td>94 ± 4</td><td>135</td><td>+ 45.4</td></tr></tbody></table>

TEM. On the contrary, for the smallest SiO₂ NPs⁻ (Φ < 50 nm), it was underestimated as compared to values provided by manufacturers and TEM. Size distributions, obtained by statistics on TEM clichés, suggest that all SiO₂ NPs are quite monodisperse (Fig. S1).

Zeta potential measurements (Table 1) indicated that, as expected, bare SiO₂ NPs⁻ were highly negatively charged at pH 5.5 resulting in the stability of the suspensions and a minimal aggregation state for these NPs. After functionalization with APS, at pH 5.5, SiO₂ NPs exhibited, as expected, highly positive charges (SiO₂ NPs⁺), also resulting in a good colloidal stability.

Thereafter, these well defined SiO₂ NPs were studied in interaction with *E. coli* cells (i) at the macroscopic level with viability tests and (ii) at the nanometer scale with AFM observations.

### 2.2. Effects of SiO₂ NPs⁻ on *E. coli* cells viability

The potential antibacterial effect of SiO₂ NPs⁻ was estimated by counting CFUs generated after treatment by a solution of NPs at a concentration of 1 g/L, on LB agar plates, and normalizing them with those of *E. coli* alone (OD = 0.01, control). Though most of such experiments required buffer solutions, ours were performed in ultra pure water for convenient reasons (AFM characterizations afterwards). No mortality is observed in this solvent (results not shown) in agreement with previous studies showing the strong resistance of *E. coli* in drastic environment, depending on pH and temperature for instance [47, 49]. The development of CFUs is known to depend on two contributions: (i) the initial distribution of the bacteria in suspension and (ii) the toxicity of the environment, i.e. its capability, after potential treatment, to generate a CFU. Thus, the influence of the presence of SiO₂ NPs⁻ is investigated under these perspectives.

Under static conditions, two sets of behavior were observed: (i) SiO₂ NPs⁻ 100 had no detrimental effect on the bacteria, with a CFU number close to those of the control, while (ii) the presence of smaller SiO₂ NPs⁻ (4 nm to 80 nm) did induce an increase in these CFUs (Fig. 1a). In addition, the clustering state of *E. coli* cells upon SiO₂ NPs⁻ treatment was studied by dark field microscopy (see Fig. 1b for representative images). In controls, *E. coli* were present as isolated cells as well as small aggregates. The same organization was observed in the presence of SiO₂ NPs⁻ 80 and 100. By contrast, in the presence of SiO₂ NPs⁻ 4, 10 and 50, the number of isolated cells was increased. Such splitting of cells clusters is likely at the origin of the CFUs increase (Fig. 1c), interactions between such SiO₂ NPs⁻ and *E. coli* being stronger than cell cell interactions. Similar behaviors were observed for *E. coli* incubated with SiO₂ NPs⁻ during 24 h, suggesting that their full activity is reached in 2 h (Fig. S2).

However, this technique appears insufficient to probe any toxicity phenomena of SiO₂ NPs⁻ as they could combine, more or less, with splitting effects (Fig. 1c).

Consequently, AFM experiments were carried out to better understand, visualize and characterize the type of interaction of SiO₂ NPs⁻ with *E. coli* and their potential deleterious effects.
<figure><img src="image_3.png" /><figcaption>Fig. 1. a) Potential antibacterial activity of SiO₂-NPs (1 g/L) against *E. coli* cells (OD = 0.01). b) Optical microscopy (dark field) observations of representative samples, with and without SiO₂-NPs, revealing modifications in cells organization and morphology (scale bar: 10 µm). c) Scheme of the potentially entangled effects of SiO₂-NPs with Φ < Φ_c and their consequences on CFUs.</figcaption></figure>

## 2.3. Morphological characterization of *E. coli* bacteria by AFM

Before investigating their interactions with SiO₂ NPs, the morphology and the structure of *E. coli* cells alone were characterized. In air, *E. coli* bacteria exhibited a typical rod shape, convention name that is widely used for such bacteria [50] (Fig. 2a). *E. coli* length, width and height were estimated, on average, at 2.0 µm, 1.2 µm and 0.3 µm, respectively. Also, *E. coli* surface showed a “brain like” aspect not only from the morphology point of view (Fig. 2b) but also from the rheology point of view with nanodomains, referred as “ripples” thereafter (Fig. 2c). The nanodomains were on average 3.9 ± 1.5 nm in height and 39.9 ± 7.4 nm in width. This particular phase separation pattern is due to the presence of an outer membrane (OM) in Gram negative bacteria. Based on the AFM tip interactions with the sample, phase contrasts on *E. coli* stand for membrane constituents with either different mechanical properties or different molecular organizations: ripples on the one hand and inter ripples on the other hand.

Morphological and structural characteristics of *E. coli* membrane being characterized, its interactions with SiO₂ NPs are hereafter studied, under these perspectives.

## 2.4. AFM study of the damages induced by SiO₂ NPs⁻ on *E. coli* cells

The potential impacts of SiO₂ NPs on the morphology and structure of *E. coli* cells are discussed hereafter, in terms of size dependency. In order to efficiently observe these damages we had to control the quantity of added SiO₂ NPs so as to image both SiO₂ NPs and bacteria the SiO₂ NPs concentration was then reduced from 1 to 0.1 g/L.

When treated with SiO₂ NPs⁻ at a concentration of 0.1 g/L, *E. coli* cells were present on the substrate as isolated cells or aggregates, as in untreated samples. All SiO₂ NPs⁻ accumulate not only around and on

bacteria but also on the substrate, as free isolated or aggregated particles (Fig. 3a b). Because of their higher stiffness, SiO₂ NPs appear as brighter zones than bacteria in phase images, which also show their respective distribution on the substrate (Fig. 3c d).

When exposed to SiO₂ NPs⁻ 80 and 100, *E. coli* cells remained rod shaped and displayed the same height as control cells (Fig. 4a b). By contrast, in the presence of SiO₂ NPs⁻ 4, 10 and 50, *E. coli* morphology evolved from rod like to more spherical shape with up to 50% decrease in their length (Fig. 4c e).

After exposure to large SiO₂ NPs⁻ 80 and 100, *E. coli* cells not only exhibited an unchanged global morphology but also an unmodified phase separation pattern of its envelop, which remained similar to that of healthy unexposed cells (Fig. 5a b and Fig. S3). As soon as smaller SiO₂ NPs⁻ (4, 10 and 50 nm) were used, strong detrimental cell membrane reorganizations were observed along with morphological changes. Indeed, the brain like structure of the OM of *E. coli* was completely altered as compared to the OM structure of unexposed cells (compare Fig. 5a,c and Fig. S3): the ripples nanodomains turned into island like structures and several areas in the OM even lack any regular organization. At this point, the reorganization might arise from either a (i) different molecular packing, i.e. the way molecules of the OM arrange themselves on the surface, or (ii) a different molecular conformation, i.e. the way molecules of the OM self orientate them selves at the air bacteria interface (condensed or fluid conformation).

SiO₂ NPs⁻ not only affect the organization of the OM but also its morphology. When Φ > 50 nm, two behaviors were observed: either (i) SiO₂ NPs⁻ were deposited on bacteria or (ii) they were trapped in between two cells (Fig. S4). In the latter case, the ripples nanodomains adapt to the NP shape, changing their orientation around the particle to partially wrap it like a “cocoon” (Fig. 6). The wrapping ratio can change among cells, leaving the NP more or less apparent (compare Fig. 6 and
<figure><img src="image_4.png" /><figcaption>Fig. 2. Characterization of *E. coli* cells morphology and rheology by AFM observations. a) Height image of the rod bumped shape of a single cell. In some cases, despite their fragility, flagella and pili are still attached to the cell (white arrow). A zoom on its irregular surface reveals (b) a typical relief and (c) a phase separation pattern corresponding to the lamellae organization of the cell OM.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 3. Height (a-b) and phase (c-d) AFM images showing the organization of *E. coli* cells treated with (a) SiO₂-NPs 100, (b) SiO₂-NPs 4, (c) SiO₂-NPs 80 and (d) SiO₂-NPs 10. NPs are either deposited on and around cells or free on the substrate (white arrows).</figcaption></figure>

Fig. S5).

When $\Phi \le 50$ nm, membrane invaginations (pore like lesions), with well defined spherical edges, were observed randomly distributed on the bacterial surface (Fig. 7a). Even if cell dependent, their depth could reach down to few tens of nanometers (Fig. S6). Two behaviors are associated to these pore like lesions: either (i) the height profile is linked to a phase signature similar to the OM one or (ii) it exhibits a

bump linked to a more repulsive signature similar to the NP one (Fig. 7b c).

We here demonstrated that drastic damages induced by SiO₂ NPs⁻ depend on a critical diameter ($50 \text{ nm} < \Phi_c < 80 \text{ nm}$) above which they were not observed anymore. The true question now is: do NPs induce any bactericidal effect?
<figure><img src="image_6.png" /><figcaption>Fig. 4. Height AFM images, emphasizing no change in the morphology of *E. coli* cells after treatment with (a) SiO₂-NPs 100, (b) SiO₂-NPs 80, and aspect ratio modification after treatment with (c) SiO₂-NPs 50, (d) SiO₂-NPs 10 and (e) SiO₂-NPs 4.</figcaption></figure>

## 2.5. Bactericidal effect of SiO₂ NPs⁻

### 2.5.1. NPs size < critical diameter Φ_c

Besides membrane degradations, the incubation of *E. coli* cells with SiO₂ NPs⁻ 4, 10 and 50 resulted, in some cases, in the collapse of *E. coli* cells. It is either partial, resulting in a drastic decrease of the bacteria height, or total, and bacteria were drained from their cellular contents and no more exhibited well organized membrane structure (Fig. 8a,b). Moreover, debris were observed on the mica substrate in the vicinity of bacteria aggregates exposed to SiO₂ NPs⁻ 4 (Fig. 8c). Such fibrous materials, already observed as the result of cell surface perturbation [51], are indicative of cellular leakages. They can also correspond to

membrane components such as LPS aggregates being expelled due to the stress induced by such NPs.

### 2.5.2. NPs size > critical diameter Φ_c

SiO₂ NPs⁻ 80 and 100 did not lead to any of these phenomena. In particular, *E. coli* cells suffered from no leakage of their intracellular compounds.

## 2.6. SiO₂ NPs charge effect on *E. coli* cells

To decorrelate between size and charge effects on the interaction between SiO₂ NPs and *E. coli*, the influence of SiO₂ NPs⁺ 100 ( > Φ_c) is

<figure><img src="image_7.png" /><figcaption>Fig. 5. AFM phase images of the OM features of *E. coli* cells (a) alone and incubated with (b) SiO₂-NPs 100 and (c) SiO₂-NPs 4 for which a transition in the ripples structures occurs.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Fig. 6. a) Height AFM image showing the wrapping of SiO₂-NPs 100 in between two neighbor cells. b) A closer view of this phenomenon in phase image. c) Profile section of a SiO₂-NPs 100 showed by the dashed line in (a). d) Proposed scheme of the OM deformation, leading to the formation of a “cocoon” around the NP.</figcaption></figure>

now investigated.

SiO₂ NPs⁺ 100 did not affect the CFUs, as for SiO₂ NPs⁻ 100. However, AFM observations emphasized the aggregation between such positive NPs and *E. coli*. Indeed, unlike SiO₂ NPs⁻ 100, SiO₂ NPs⁺ 100 tended to exclusively accumulate and stick to, around and on bacteria, none being observed as free isolated or aggregated particles on the substrate (compare Fig. 3 and Fig. 9a). This emphasizes the privileged electrostatic affinity between SiO₂ NPs⁺ and the negatively charged surface of *E. coli*, as confirmed by zeta potential measurements.

Furthermore, modifying the charge of SiO₂ NPs 100 did not induce any change in *E. coli* rod shape morphology (Fig. 9b) nor in its OM ripples structure (Fig. 9c). However, unlike SiO₂ NPs⁻ 100 which did not lead to any membrane degradations, SiO₂ NPs⁺ 100 induced drastic damages including the formation of (i) extra membrane aggrec gates and (ii) membrane invaginations. In the first case, aggregates with heights ranging from 4 to 15 nm surrounded cells (Fig. 10a b). In the second case, pore like lesions, similar to those observed with SiO₂ NPs⁻ smaller than Φₑ, exhibited a depth of few tens of nanometer, which are not correlated to the NP size (Fig. 10c d).

### 3. Discussion

This work is the first study of exposure to NPs of the strain *E. coli* MRE 162, which was used, during the 70's, for military purposes. Although thought to be inoffensive, these *E. coli* were reported to cause diseases in highly susceptible individuals. This emphasizes the necessity of investigating on proper antibacterial agents (harmless for human cells) such as NPs, for commonly found bacteria like *E. coli*.

Former studies on the toxicity of SiO₂ NPs present contradictory results not only because of different methodologies but also because they do not necessarily address the same size nor the same surface state of SiO₂ NPs, without explicitly mentioning these parameters in some cases. It is thus difficult to compare these results and to potentially

demonstrate a size and charge effect tendency. In addition, despite a well known dose dependent cytotoxicity of antibacterial agents such as NPs [52], these studies differ in terms of concentration: *E. coli* growth inhibition was reported for a dose exposure of 2.0 g/L [38,53] while, in another study, a concentration as low as 0.2 g/L was inefficient [54].

Consequently, we undertook a systematic approach of this issue. We report herein (i) the potential antibacterial activity of SiO₂ NPs⁻, with diameters varying from 4 to 100 nm, and (ii) the influence of the surface charge for the largest SiO₂ NPs (SiO₂ NPs⁺ 100) on *E. coli* cells. Based on previous studies and submitted to AFM observation conditions (to correctly visualize both NPs and bacteria), toxicity tests were performed, under static conditions, after a 2 h incubation with NPs at a concentration of 0.1 g/L. Their toxicity was rationalized with regards to their size and charge effects.

Viability experiments revealed the existence of a NP size threshold, 50 nm < Φₑ < 80 nm. Above Φₑ, SiO₂ NPs⁻ had no influence on CFUs nor on the organization of *E. coli* population. Below Φₑ, SiO₂ NPs⁻ induced an increase in CFUs associated to the easier separation of the initial cell clusters. This phenomenon is not due to an improved ability of cells to divide but rather to the capacity of these SiO₂ NPs⁻, despite their negative charge, to interact with the LPS layer of the bacteria OM, probably through hydrogen bonding with the LPS hydroxyl groups. These interactions are likely stronger than cell cell ones as they could break down cells clusters. To the best of our knowledge, this splitting effect has never been reported nor correlated to a size dependent action of NPs on cells. Former studies only described the size effect of NPs in terms of CFUs decrease always related to viability losses: for instance, the antibacterial activity decreases with an increase of the size of TiO₂ [31] and Ag [55] NPs. We, here, emphasized that CFUs counting is definitely insufficient by itself to probe toxicity phenomena since it reflects a competition between (i) the number of isolated cells, increased by splitting effects and (ii) the number of viable cells, decreased by potential antibacterial
<figure><img src="image_9.png" /><figcaption>Fig. 7. a) AFM amplitude image showing membrane invaginations (white arrows) when E. coli cells are exposed to SiO₂-NPs. b) AFM phase image of a closer view of membrane invaginations with SiO₂-NPs. c) and d) corresponding height profiles of the pore-like lesions pointed by red and blue lines in (b). Proposed scheme of the OM deformation is also shown in (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Fig. 8. Bactericidal effects of SiO₂-NPs with Φ < Φ_c against E. coli cells. AFM height images of partially and totally collapsed cells when interacting with (a) SiO₂-NPs 50 and (b) SiO₂-NPs 10. c) AFM amplitude image showing debris due to bacteria lysis on the substrate when E. coli cells interact with SiO₂-NPs. d) Damages are indicated by full arrows.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Fig. 9. a) AFM height images of *E. coli* cells population with SiO₂-NPs⁺ 100, aggregated on cells (white arrow). AFM height (b) and phase (c) images of *E. coli* incubated with SiO₂-NPs⁺ 100 showing its unchanged morphology and OM structure.</figcaption></figure>

activity (Fig. 2c).

From this point of view, AFM proved to provide complementary and
necessary information, at the cellular level, on the NPs size dependent
antibacterial activity. Firstly, it emphasized the particular ultrastruc-
ture present on healthy *E.coli*'s surface. This “brain like” organization,

here called ripples, is attributed to the conformation of LPS molecules
in the OM [56]. Then, AFM observations confirmed, at the cellular
level, the existence of a NPs size threshold, 50 nm < Φ<sub>c</sub> < 80 nm. On
the one hand, SiO<sub>2</sub> NPs<sup>-</sup> larger than Φ<sub>c</sub> do not induce any particular
damage on the overall morphology of E. coli nor on its OM structure.

<figure><img src="image_12.png" /><figcaption>Fig. 10. *E. coli* membrane damages induced by SiO₂-NPs⁺ 100, observed on AFM phase (a) and amplitude (c) images, and pointed by full arrows. a) Formation of extra-aggregates, with (b) height profiles representative of diverse aggregates. c) Formation of membrane invaginations with (d) height profiles representative of diverse invaginations.</figcaption></figure>
Noteworthy is the partial wrapping observed for these NPs trapped in between cells (Fig. 6c). A balance between adhesion and free energies is here assumed for the NP not to translocate through cells, as already observed by Livadaru and Kovalenko in a statistical mechanical approach of membrane invaginations [57]. On the other hand, SiO₂ NPs⁻ smaller than Φ_c strongly disturb *E. coli* cells. The pore like lesions observed are deep enough, as compared to the OM thickness (around 8 nm), for these SiO₂ NPs⁻ to (i) disrupt this OM and induce its invagination or piercing and even to (ii) damage the underlying peptidoglycan layer (Fig. 7c) which is involved not only in cell division processes but also in the preservation of the cell wall uniformity and cell shape. Permeabilization of this layer is also reinforced by the more spherical shape of bacteria. Such invaginations were reported in antibiotic (penicillin and amoxicillin) treatment of *E. coli*, interfering with enzymes involved in the peptidoglycan synthesis [58]. In addition, SiO₂ NPs⁻ smaller than Φ_c induce a reorganization in *E. coli*'s OM structure from a ripples pattern to more compact and rounded aggregates, typical of weakened bacteria [56]. This transition might arise from (i) the spatial reorganization of the LPS molecules from a ripples structure to an aggregated one or (ii) from the transition in their molecular conformation from an extended to a more condensed one. In both cases, this transition is in agreement with the polymer like behavior [59] of LPS when undergoing an external pressure [60]. It is likely due to small SiO₂ NPs⁻ interfering in the core of the OM and forcing the molecules to reorganize as compression does in the case of Langmuir monolayer experiments (Fig. S3) [60]. It is not excluded either that a strong hydrogen bonding between these SiO₂ NPs⁻ and the multiple hydroxyls groups of LPS molecules does play a role in this disorganization. It induces a break in the crystalline organization of LPS and thus the loss of the brain like structure, this process being much easier with smaller NPs. At some point, especially for the 4 and 10 nm sizes, NPs might penetrate in the grooves of the LPS layer. Finally, observed lysis and consequent leakage of intracellular components, could occur through these pore like lesions or through the complete and direct destruction of the cell inner membrane. Indeed, cell lysis suggests that, when SiO₂ NPs⁻ reach the peptidoglycan layer, they are intercalated and translocated, and reach the inner membrane leading to potential toxicity. For cells remaining alive (healthy appearance), SiO₂ NPs⁻ might be trapped outside the peptidoglycan layer protecting the inner membrane and thus the cell integrity. Considering these damages, SiO₂ NPs⁻ smaller than Φ_c have similar effects as some antimicrobial peptides following the “carpet model” [61,62].

Independently of their sizes, the positive surface charge of SiO₂ NPs generates strong attractive electrostatic interactions with negatively charged *E. coli* bacteria. This affinity appears to favor antibacterial activity of SiO₂ NPs. Indeed, SiO₂ NPs⁺ 100 lead to drastic membrane invaginations, similar to those observed with SiO₂ NPs⁻ smaller than Φ_c and blebbing phenomenon. As Φ > Φ_c, the size influence is, here, ruled out, implying a different mechanism for such damages, probably a consequence of the electrostatic field induced by SiO₂ NPs⁺. This surface charge dependent toxicity confirms previous studies on silver NPs either bare or coated [30,63].

## 4. Conclusion

We have demonstrated the size and charge dependent toxicity of SiO₂ NPs towards *E. coli* bacteria, through a systematic approach. Cells were exposed to commercial and home made SiO₂ NPs with different sizes (4, 10, 50, 80 and 100 nm), either bare (SiO₂ NPs⁻) or functionalized (SiO₂ NPs⁺) and their toxicity emphasized by viability tests and AFM observations. We demonstrated the existence of a critical diameter, 50 nm < Φ_c < 80 nm, under which cell damages were observed: OM invaginations and reorganization (from ripples for healthy cells to aggregates for weakened and damaged ones). We reported, for the first time, the splitting effects of these SiO₂ NPs⁻ on cells and its potential interaction with toxicity phenomena. We also showed that

SiO₂ NPs larger than Φ_c did not affect *E. coli* cells unless they were positively charged, SiO₂ NPs⁺ 100 inducing similar damage as the smallest negative SiO₂ NPs⁻. Finally, the particular membrane reorganization emphasized the importance of LPS structure in *E. coli* in the resistance to external stress and the need for further studies on its nanomechanical properties and its interaction with NPs.

## 5. Materials and methods

For convenient reasons, we will use thereafter short names for all used NPs. For instance, negatively charged silica NPs of 4 nm in diameter will stand as SiO₂ NPs⁻ 4. Similar expressions will be used for all NPs.

### 5.1. Materials

Commercial SiO₂ NPs⁻ 10, 50 and 100 were purchased from Biovalley (Marne la Vallée, France) and SiO₂ NPs⁻ 4 from Alfa Aesar (Schiltigheim, France). Gram negative *E. coli* bacteria (MRE 162 strain) were a kind gift from the Centre d'Etudes du Bouchet, DGA (Direction Générale de l'Armement, France). Mica, purchased from Electron Microscopy Sciences (Hatfield, United States), was used as support for all AFM measurements. Luria Broth (LB) medium was used as nutritive medium and purchased from Fisher Scientific (Waltham, Massachusetts, United States). L-Arginine and tetraethylorthosilicate (TEOS), used for silica seeds synthesis, were purchased from Sigma Aldrich (Saint Louis, Missouri, United States) as well as aminopropyl triethoxysilane (APS), used for NPs functionalization. Ethanol and ammonium hydroxide, used for SiO₂ NPs growth, were also purchased from Sigma Aldrich. Millipore ultrapure water (pH 5.5, resistivity > 18.2 MΩ cm) was used for NPs cleaning and bacteria suspensions preparation.

### 5.2. Preparation and characterization of SiO₂ NPs solutions

To obtain a large range of NPs sizes, we used both commercial and home synthesized SiO₂ NPs. Home synthesis was also used to functionalize the negatively charged NPs (SiO₂ NPs⁻) and get a positive surface charge (SiO₂ NPs⁺).

Commercial SiO₂ NPs⁻ were centrifuged three times at 9000g for 45 min (20 min were enough for SiO₂ NPs⁻ 100), cleaned and redispersed in ultrapure water. SiO₂ NPs⁻ were then placed in a sonication bath for approximately 1 h to ensure minimal aggregation in the solution.

Synthesis of home made SiO₂ NPs⁻ was carried out following synthesis developed by Hartlen et al. [64]. The first step consisted in synthesizing SiO₂ seeds. 100 mL of a 6 mM L-arginine water solution was introduced in a vial at 60 °C, under magnetic stirring (150 rpm). After temperature stabilisation, 10 mL of TEOS were slowly added so as to respect the interface. Seeds of about 30 nm in diameter were obtained after complete consumption of TEOS. The second step consisted in the growth of these seeds. Typically, 10 mL of SiO₂ seeds were introduced in a mixture of 455 mL of ethanol and 35 mL of ammonium hydroxide in water solution ([NH₃] = 1 M), under stirring (700 rpm). TEOS was then introduced in the amount calculated to get the targeted diameter, at a rate of 0.5 mL/h [65]. Surface modification was performed by adding APS (six equivalents assuming a rate of 2 3 µmol/m² of active OH groups at the surface of the silica particles) to the SiO₂ NPs⁻ solution; the mixture was then stirred for 48 h in a water bath at room temperature, and then for 2 h at 100 °C. Final SiO₂ NPs⁺ were washed using ultrapure water and ethanol and 3 cycles of centrifugation (13,000 rpm for 20 min.)

Dynamic Light Scattering (DLS) experiments were carried out on a Malvern Zetasizer Nano ZS setup (Orsay, France) to determine the size and polydispersity of NPs. This method provides information on both the hydrodynamic diameter of the SiO₂ NPs in solution and their
polydispersity. Transmission Electron Microscopy (TEM) with a JEOL JEM 1400 Plus and AFM observations were also used to investigate not only the size and morphology of SiO₂ NPs but also their organization on a substrate. Consequently, while TEM images give the silica NPs diameter (i.e. its core), DLS measurements give the hydrodynamic diameter of the NPs including the first sphere of coordination of the solvent. Zeta potential measurements were carried out on a Wallis Zeta potential analyzer from Cordouan (Pessac, France). For these three characterizations, SiO₂ NPs were suspended in ultrapure water to obtain a final concentration of 0.1 g/L and sonicated before any experiments.

### 5.3. Bactericidal susceptibility test

A fresh *E. coli* culture was obtained by inoculating 50 mL of LB medium with 250 µL of an overnight culture. The bacterial growth was followed by measuring the optical density (OD) at 600 nm, using a Helios gamma spectrophotometer (Thermo Scientific), until it reached 0.60. The suspension was further centrifuged (8000 rpm for 10 min. at 4 °C), and the cell pellet was re diluted in ultrapure water to obtain an OD of 0.01. Finally, NPs were introduced in the suspension at the desired concentration (1 g/L) and left to interact for 2 h, under static conditions, at room temperature. At the end of the incubation period, ten fold serial dilutions in ultrapure and sterile water of the suspensions were made and 100 µL of each dilution were spread onto LB agar plates. Bactericidal effects were evaluated by counting the number of colonies (CFU for colony forming units) developed on plates after 16 h at 37 °C. Average values of three independent experiments are reported there after.

### 5.4. Dark field microscopy observations

For dark field microscopy observations, *E. coli* cells were grown in LB medium until the culture reached an OD = 0.60 at 600 nm. The bacterial cells were centrifuged (8000 rpm for 10 min at 4 °C) and re suspended in ultrapure, sterile water to obtain an OD = 0.60 at 600 nm before being incubated in the presence of the different NPs at 1 g/L for 2 h at room temperature under static conditions. A microscope Eclipse Ni (Nikon) equipped with a dark field condenser (oil 1.463 1.20) and an objective Achromat 40 × /0.65 both from Nikon was used for dark field image acquisition. Images were acquired with a Nikon DS Qi1Mc camera and NIS Elements software. Representative images are provided thereafter.

### 5.5. Preparation of NPs *E. coli* suspensions for AFM experiments

*E. coli* cells were grown on LB agar plates and incubated for 16 h at 37 °C. After scraping, bacteria were suspended in ultrapure water and their concentration was adjusted to 10⁶ cells/mL. An equivalent volume of *E. coli* cells and SiO₂ NPs suspensions at a concentration of 0.1 g/L, were mixed and gently manually shaken (4 times every 20 min), then left to interact at room temperature for 2 h. Five microliters of each mixed suspension was deposited on freshly cleaved mica, left to dry in a desiccator and imaged by AFM the following day.

### 5.6. AFM experiments

Briefly, AFM consists of a cantilever with a nanoprobe at its end which, along with a piezo scanner, scans the sample surface. In close proximity, small (attractive and repulsive) forces between the probe and the surface lead to the deflection of the cantilever, which is recorded. This technique gives information on morphological and mechanical properties (adhesion, visco elasticity) of a surface, at the nanometer level. In this work, AFM experiments were carried out using a Bioscope II mounted on an IX71 Olympus inverted optical microscope operating with the NanoScope V controller and also with a Multimode

AFM (Veeco Brucker, Santa Barbara, USA).

Though, imaging in liquid is preferred for cells hydration, air conditions were privileged for a better resolution [42,66] (due to tip bacteria interactions), notably on the topographical and nanomechani cal contrasts on the surface structure of bacteria. It also allows to work in dynamic tapping mode and, consequently, to take advantage of the harmonic oscillator behavior of the cantilever. Thus, very small forces were used, preventing any damages to the samples. Tapping mode images were obtained with commercial cantilevers with a spring constant around 40 N/m, at a scan rate of 0.5 to 1.0 Hz and with a resolution of 512 × 512 pixels. For each experiment, height, amplitude and trace phase images were recorded. Height images stand for topographical relief of the sample. Phase images show the phase difference between exciting and response signals, arising from the sample nanomechanical properties. Thus, they are linked to (i) dissipa tion processes between the tip and the sample and (ii) surface hardness. For instance, a homogenous surface (composition and organization) will not show any significant contrast in phase. Amplitude images (error signal) provide additional information on the topography and morphology of very fine details which might be saturated in height images.

Hereafter, samples were observed, in tapping mode, in the day following their preparation to prevent any interference, like ageing of *E. coli* biofilm [56], with the potential damage caused by SiO₂ NPs. Different areas were systematically scanned and images shown there after are representative of the cells in a given set of colonies and of all the tested samples.

## Acknowledgments

The authors thank the Région Aquitaine and CNRS (France) for supporting this work through the equipment of the NanoSpectroImagerie (NSI LOMA) platform used in this work (CPER COLA2). They are grateful to the Direction Générale de l'Armement (DGA, Ministère de la Défense France, grant number : 2014017) and the Région Aquitaine (France) for their financial support through the Ph.D. grant of M. Mathelié Guinlet. Finally, authors thank NSI LOMA plat form for technical help and J.P Chapel for its helpful advises and discussions about DLS measurements.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.cis.2017.04.012.

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