Jazia Awassa

Damien Cornu
email: damien.cornu@univ-lorraine.fr

Christian Ruby

Sofiane El-Kirat-Chatel

of reactive oxygen species: How to optimize the antibacterial effects of layered double hydroxides

Keywords:

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Infectious diseases caused by microbial contamination are threatening human health all over the world [START_REF] Fitzpatrick | Modelling microbial infection to address global health challenges[END_REF]. Infections by pathogenic microorganisms are of serious concern to several fields such as food industries [START_REF] Chatterjee | Microbial Contamination, Prevention, and Early Detection in Food Industry[END_REF], textile industries [START_REF] Md | Microorganisms, infection and the role of medical textiles[END_REF], healthcare [START_REF] Laheij | Healthcare-associated viral and bacterial infections in dentistry[END_REF], and pharmaceutical products [START_REF] Eissa | Distribution of bacterial contamination in non-sterile pharmaceutical materials and assessment of its risk to the health of the final consumers quantitatively, Beni-Suef[END_REF].

Traditional antibiotic drugs can usually treat bacterial infections, however, their extensive use had led to bacterial resistance and the spread of resistant strains [START_REF] Christaki | Antimicrobial Resistance in Bacteria: Mechanisms, Evolution, and Persistence[END_REF][START_REF] Nathan | Resisting antimicrobial resistance[END_REF]. Recent advances in the field of material science had opened up new gates for designing new antimicrobials based on synthetic inorganic materials [START_REF] Forano | Interactions between Biological Cells and Layered Double Hydroxides: Towards Functional Materials[END_REF][START_REF] Häffner | Membrane interactions and antimicrobial effects of inorganic nanoparticles[END_REF][START_REF] Wang | Advances in Antimicrobial Organic and Inorganic Nanocompounds in Biomedicine[END_REF] In fact, inorganic antibacterial materials are considered advantageous over organic antibacterial materials due to their higher stability, recyclability and improved safety [START_REF] Kumar | Antimicrobial properties of ZnO nanomaterials: A review[END_REF].

Among the various inorganic materials, layered double hydroxides (LDHs) were extensively studied due to their interesting properties as anion exchangeability, compositional flexibility, and biocompatibility, thus making them good candidates for several potential applications as catalysis [START_REF] Kim | Ternary Layered Double Hydroxides (LDHs) Based on Co-, Cu-Substituted ZnAl for the Design of Efficient Photocatalysts: Ternary Layered Double Hydroxides (LDHs) Based on Co-, Cu-Substituted ZnAl for the Design of Efficient Photocatalysts[END_REF][START_REF] Razzaq | Layered Double Hydroxide (LDH) Based Photocatalysts: An Outstanding Strategy for Efficient Photocatalytic CO2 Conversion[END_REF], sorption of pollutants [START_REF] Johnston | Understanding Layered Double Hydroxide properties as sorbent materials for removing organic pollutants from environmental waters[END_REF][START_REF] Williams | Removal of copper from cattle footbath wastewater with layered double hydroxide adsorbents as a route to antimicrobial resistance mitigation on dairy farms[END_REF], biosensors [START_REF] Yang | A novel fluorescent DNA sensor system based on polydopamine modified MgAl-layered double hydroxides[END_REF], drug storage delivery [START_REF] Bi | Layered Double Hydroxide-Based Nanocarriers for Drug Delivery[END_REF][START_REF] Senapati | Layered double hydroxides as effective carrier for anticancer drugs and tailoring of release rate through interlayer anions[END_REF], and antimicrobial materials [START_REF] Kim | Ternary Layered Double Hydroxides (LDHs) Based on Co-, Cu-Substituted ZnAl for the Design of Efficient Photocatalysts: Ternary Layered Double Hydroxides (LDHs) Based on Co-, Cu-Substituted ZnAl for the Design of Efficient Photocatalysts[END_REF][START_REF] Ballarin | Hybrid material based on ZnAl hydrotalcite and silver nanoparticles for deodorant formulation[END_REF][START_REF] Donnadio | Bioinspired Reactive Interfaces Based on Layered Double Hydroxides-Zn Rich Hydroxyapatite with Antibacterial Activity[END_REF][START_REF] Li | High and long-term antibacterial activity against Escherichia coli via synergy between the antibiotic penicillin G and its carrier ZnAl layered double hydroxide, Colloids Surf[END_REF][START_REF] Moaty | Preparation, characterization and antimicrobial applications of Zn-Fe LDH against MRSA[END_REF]. Layered double hydroxides (LDHs), also known as anionic clays, are lamellar solids having a general formula expressed as [M(II)(1-x) M(III)x (OH)2] x+ [A n- x/n

. yH2O] x-, where M(II) and M(III) represent divalent and trivalent metallic cations respectively, and A n-is an exchangeable intercalating anion. The LDH structure (Fig. 1A) is based on the stacking of positively charged brucite-like sheets comprising the divalent and trivalent cations linked by OH units coordinated at the octahedral positions forming sheets, which are then stacked up on top of each other to yield a layered structure similar to that of brucite mineral [Mg(OH)2]

[ [START_REF] Mohapatra | A review on the recent progress, challenges and perspective of layered double hydroxides as promising photocatalysts[END_REF][START_REF] Nalawade | Layered double hydroxides: A review, Layer[END_REF][START_REF] Rives | Layered double hydroxides: Present and Future[END_REF][START_REF] Theiss | Synthesis of layered double hydroxides containing Mg2+, Zn2+, Ca2+ and Al3+ layer cations by co-precipitation methods-A review[END_REF][START_REF] Cavani | Hydrotalcite-type anionic clays: Preparation, properties and applications[END_REF]. These brucite-like sheets hold a net positive charge which is compensated by negatively charged anions along with water molecules in the interlaminar regions. LDH-based materials were utilized for several biological applications especially antimicrobial ones [START_REF] Forano | Interactions between Biological Cells and Layered Double Hydroxides: Towards Functional Materials[END_REF][START_REF] Allou | Hybrid nanocomposites of layered double hydroxides: an update of their biological applications and future prospects[END_REF][START_REF] Yan | Layered double hydroxide nanostructures and nanocomposites for biomedical applications[END_REF]. In fact, several surveys showed that LDHs possess high potential as carriers of functional antibacterial molecules as antibiotics because of their high biocompatibility, high chemical stability, and controlled release rate [START_REF] Khan | Controlled release of organic–inorganic nanohybrid:cefadroxil intercalated Zn–Al-layered double hydroxide[END_REF][START_REF] Mishra | Orientation of Organic Anions in Zn-Al Layered Double Hydroxides with Enhanced Antibacterial Property[END_REF][START_REF] Wang | Synthesis, characterization, and controlled release antibacterial behavior of antibiotic intercalated Mg-Al layered double hydroxides[END_REF]. In addition, LDH can also be loaded with nanoparticles (NPs) such as gold, silver, copper and zinc oxide NPs which are known to treat several diseases caused by bacterial infections [START_REF] Ballarin | Hybrid material based on ZnAl hydrotalcite and silver nanoparticles for deodorant formulation[END_REF][START_REF] Bogdanović | Copper nanoparticles with high antimicrobial activity[END_REF][START_REF] Dakal | Mechanistic Basis of Antimicrobial Actions of Silver Nanoparticles[END_REF][START_REF] Mohamed | Antibacterial effect of gold nanoparticles against Corynebacterium pseudotuberculosis[END_REF][START_REF] Pati | Topical application of zinc oxide nanoparticles reduces bacterial skin infection in mice and exhibits antibacterial activity by inducing oxidative stress response and cell membrane disintegration in macrophages[END_REF][START_REF] Ma | Core-Shell Gold Nanorod@Layered Double Hydroxide Nanomaterial with Highly Efficient Photothermal Conversion and Its Application in Antibacterial and Tumor Therapy[END_REF]. The assembly of metallic NPs-in inorganic matrices as of LDHs can provide an inexpensive and biocompatible means to help limit the problem of aggregation and high exposure dose of free NPs [START_REF] Carja | Nanosized silver-anionic clay matrix as nanostructured ensembles with antimicrobial activity[END_REF]. Moreover, recent studies showed that even pristine LDHs, more precisely Zn and Cu-based pristine LDHs, could induce an efficient antibacterial without any further modification [START_REF] Moaty | Preparation, characterization and antimicrobial applications of Zn-Fe LDH against MRSA[END_REF][START_REF] Dutta | Zn 2 Al-CO 3 Layered Double Hydroxide: Adsorption, Cytotoxicity and Antibacterial Performances[END_REF][START_REF] Li | Efficient antimicrobial properties of layered double hydroxide assembled with transition metals via a facile preparation method[END_REF][START_REF] Tabti | Facile synthesis of Cu-LDH with different Cu/Al molar ratios: application as antibacterial inhibitors[END_REF][START_REF] Awassa | Divalent metal release and antimicrobial effects of layered double hydroxides[END_REF].

Each type of antibacterial LDH, e.g. antibacterial molecules-loaded LDHs, NPs-loaded LDHs and pristine LDHs, may induce a different antibacterial mode of action. Fig. 1B illustrates the three different suggested mechanisms explaining the antimicrobial activity of these different types of antibacterial LDHs: [START_REF] Fitzpatrick | Modelling microbial infection to address global health challenges[END_REF] direct surface interactions between pristine and NPs-loaded LDHs with the bacteria thus leading to their growth inhibition or death [START_REF] Carja | Nanosized silver-anionic clay matrix as nanostructured ensembles with antimicrobial activity[END_REF][START_REF] Dutta | Zn 2 Al-CO 3 Layered Double Hydroxide: Adsorption, Cytotoxicity and Antibacterial Performances[END_REF][START_REF] Tabti | Facile synthesis of Cu-LDH with different Cu/Al molar ratios: application as antibacterial inhibitors[END_REF][START_REF] Mishra | Antibacterial actions of silver nanoparticles incorporated Zn-Al layered double hydroxide and its spinel[END_REF][START_REF] Zhang | Colloidal Surface Engineering: Growth of Layered Double Hydroxides with Intrinsic Oxidase-Mimicking Activities to Fight Against Bacterial Infection in Wound Healing[END_REF], [START_REF] Chatterjee | Microbial Contamination, Prevention, and Early Detection in Food Industry[END_REF] dissolution by through the controlled release of antibacterial molecules present in the LDH interlaminar region of antibacterial molecules-loaded LDH composites [START_REF] Djebbi | Delivery system for berberine chloride based on the nanocarrier ZnAllayered double hydroxide: Physicochemical characterization, release behavior and evaluation of anti-bacterial potential[END_REF][START_REF] Ryu | Layered double hydroxide as novel antibacterial drug delivery system[END_REF], or through the release of release constituent metallic ions, e.g. Zn 2+ , Cu 2+ , Ag + from NPs-loaded and pristine LDHs [START_REF] Kim | Ternary Layered Double Hydroxides (LDHs) Based on Co-, Cu-Substituted ZnAl for the Design of Efficient Photocatalysts: Ternary Layered Double Hydroxides (LDHs) Based on Co-, Cu-Substituted ZnAl for the Design of Efficient Photocatalysts[END_REF][START_REF] Dutta | Zn 2 Al-CO 3 Layered Double Hydroxide: Adsorption, Cytotoxicity and Antibacterial Performances[END_REF][START_REF] Awassa | Divalent metal release and antimicrobial effects of layered double hydroxides[END_REF][START_REF] Nocchetti | Ag/AgCl nanoparticle decorated layered double hydroxides: synthesis, characterization and antimicrobial properties[END_REF],

(3) generation of reactive oxygen species (ROS) by pristine and NPs-loaded LDHs to yield an efficient antibacterial effect [START_REF] Yang | A novel fluorescent DNA sensor system based on polydopamine modified MgAl-layered double hydroxides[END_REF][START_REF] Ma | Core-Shell Gold Nanorod@Layered Double Hydroxide Nanomaterial with Highly Efficient Photothermal Conversion and Its Application in Antibacterial and Tumor Therapy[END_REF][START_REF] Dutta | Zn 2 Al-CO 3 Layered Double Hydroxide: Adsorption, Cytotoxicity and Antibacterial Performances[END_REF][START_REF] Li | Efficient antimicrobial properties of layered double hydroxide assembled with transition metals via a facile preparation method[END_REF][START_REF] Tabti | Facile synthesis of Cu-LDH with different Cu/Al molar ratios: application as antibacterial inhibitors[END_REF]49]. Table 1 reports the antibacterial performance of pristine and NPs-loaded LDHs together with the suggested antibacterial mechanisms provided in previous literature.

In fact, numerous reviews reported the synthesis routes of LDH materials and their various applications [START_REF] Bukhtiyarova | A review on effect of synthesis conditions on the formation of layered double hydroxides[END_REF][START_REF] Chaillot | Layered double hydroxides and LDH-derived materials in chosen environmental applications: a review[END_REF][START_REF] Chubar | Layered double hydroxides as the next generation inorganic anion exchangers: Synthetic methods versus applicability[END_REF][START_REF] Wang | Recent Advances in the Synthesis and Application of Layered Double Hydroxide (LDH) Nanosheets[END_REF], however, only a few focused on their antimicrobial effects [START_REF] Forano | Interactions between Biological Cells and Layered Double Hydroxides: Towards Functional Materials[END_REF][START_REF] Mishra | Layered double hydroxides: A brief review from fundamentals to application as evolving biomaterials[END_REF][START_REF] Rives | Intercalation of drugs in layered double hydroxides and their controlled release: A review[END_REF][START_REF] Sharma | Layered double hydroxide as multifunctional materials for environmental remediation: from chemical pollutants to microorganisms[END_REF].

Although these reviews explained the efficiency of LDHs to serve as antimicrobial materials, the information given concerned more generally different types of biological applications and not specifically the antimicrobial ones. Moreover, none of these reviews reported in detail the different antibacterial mechanisms occurring at the biointerface between LDHs and bacteria.

In this review, several suggested antibacterial mechanisms of the different types of antibacterial LDHs are discussed in three sections. Of course, it is not possible to isolate only one antibacterial mechanism, because some antimicrobial effects of LDHs result from the combination of direct contact, ion or antimicrobial molecules release and ROS generation. However, some specificities can be correlated to the different suggested mechanisms. For instance, the long-distance effect through molecules or ion release, or the effect of light for the ROS generation. Therefore, each section starts with different physico-chemical considerations, followed by various experimental observations of these mechanisms and is concluded with some considerations on how to tune the properties of LDHs to maximize the antimicrobial effect through the suggested mechanism.

LDH antibacterial activity by direct surface interactions

2.1.Electrostatic interactions between bacteria and LDHs

From a physicochemical point of view, microorganisms as bacteria can be considered as living colloidal particles. This is due to their compositional polypeptides having ionizable weakly acidic and basic groups, e.g. carboxyl and amino groups [START_REF] Harden | THE ISOELECTRIC POINT OF BACTERIAL CELLS[END_REF][START_REF] Wilson | Status of methods for assessing bacterial cell surface charge properties based on zeta potential measurements[END_REF]. Therefore, materials possessing positively charged surfaces are believed to be ideal nuclei for microbial adhesion [START_REF] Gottenbos | Antimicrobial effects of positively charged surfaces on adhering Gram-positive and Gram-negative bacteria[END_REF]. LDHs have a positively charged surface with zeta potential values up to 30-50 mV [START_REF] Xu | Stable Suspension of Layered Double Hydroxide Nanoparticles in Aqueous Solution[END_REF], large surface areas of 10-200 m 2 /g [START_REF] Liu | Adsorption of bacteria onto layered double hydroxide particles to form biogranule-like aggregates[END_REF], and a high anion exchange capacity of 2-5 mmol/g [START_REF] Miyata | Anion-Exchange Properties of Hydrotalcite-Like Compounds[END_REF], thus meeting all the criteria for an ideal bacterial adhesion.

Jin et al. reported the removal of Gram-negative bacteria Escherichia coli by chloride-intercalated pristine MgAl and ZnAl LDHs for water purification applications [START_REF] Jin | Removal of bacteria and viruses from waters using layered double hydroxide nanocomposites[END_REF]. LDH slurry systems showed a bacterial adsorption efficiency ≥ 99%. Similarly, the flow-through column showed an adsorption efficiency as high as 87 to 99%. Such findings suggested that LDHs could potentially be used as a sorbent material to purify untreated water from bacteria. 2. The first suggested interaction involved electrostatic interactions between the negatively charged bacterial membrane (due to the presence of PO4 -groups) and the positively charged LDH surface (Region A, Fig. 2). Bacterial adsorption could also be enhanced by anion exchange reactions, where PO4 -groups present at the surface of the bacterium could gradually replace individual CO3 2-on the LDH surface (Region B, Fig. 2). Bacterial adsorption could be further achieved by the substitution of the OH groups on the LDH surface with the PO4 -groups (Region C, Fig. 2). The last suggested adhesion mechanism was that PO4 -groups present on the bacterial surface and the OH groups on the LDH surface would interact with each other via hydrogen bonds and/or polar attractions (Region D, Fig. 2). These mentioned investigations did not correlate the adsorption of bacteria on LDHs to their antibacterial activity where no antibacterial activity tests were carried out.

Topological adhesion of LDH on bacteria

One other possible reason explaining LDH-bacteria adhesion is the rough surface of LDHs thus promoting the adhesion phenomena which may be a key factor contributing to their antibacterial activity [START_REF] Zhang | Colloidal Surface Engineering: Growth of Layered Double Hydroxides with Intrinsic Oxidase-Mimicking Activities to Fight Against Bacterial Infection in Wound Healing[END_REF][START_REF] Wang | Flower-like Surface of Three-Metal-Component Layered Double Hydroxide Composites for Improved Antibacterial Activity of Lysozyme[END_REF]. It had been well known that the motility of bacteria is essential for bacterial normal functions [START_REF] Tamar | The role of motility and chemotaxis in the bacterial colonization of protected surfaces[END_REF], and that rough antimicrobial colloidal surfaces might adhere to bacteria and disturb its normal motility functions [START_REF] Song | Silica Nanopollens Enhance Adhesion for Long-Term Bacterial Inhibition[END_REF][START_REF] Yan | Atomic-engineered gold@silvergold alloy nanoflowers for in vivo inhibition of bacteria[END_REF]. Zhang et al. successfully synthesized antibacterial hybrids of silica nanorods and unsmooth hollow Mn/Ni LDHs [START_REF] Zhang | Colloidal Surface Engineering: Growth of Layered Double Hydroxides with Intrinsic Oxidase-Mimicking Activities to Fight Against Bacterial Infection in Wound Healing[END_REF]. The antibacterial activities of SiO2, Mn/Ni LDHs and SiO2 nanorods @Mn/Ni LDHs were investigated against Gram-negative E. coli LDH providing better contact with bacteria and thus an amplified antibacterial effect [START_REF] Wang | Flower-like Surface of Three-Metal-Component Layered Double Hydroxide Composites for Improved Antibacterial Activity of Lysozyme[END_REF]. LDHs adhesion to bacteria could further affect the bacterial biological activities, such as blocking the channels for nutrients uptake and waste exclusion, which would subsequently lead to bacterial death or inhibition [START_REF] Li | High and long-term antibacterial activity against Escherichia coli via synergy between the antibiotic penicillin G and its carrier ZnAl layered double hydroxide, Colloids Surf[END_REF][START_REF] Ryu | Layered double hydroxide as novel antibacterial drug delivery system[END_REF][START_REF] Peng | The prospect of layered double hydroxide as bone implants: A study of mechanical properties, cytocompatibility and antibacterial activity[END_REF]. Moreover, LDHs may also adsorb many essential nutrients for bacterial growth in medium, e.g. proteins, celluloses, starch and proteins [START_REF] Li | High and long-term antibacterial activity against Escherichia coli via synergy between the antibiotic penicillin G and its carrier ZnAl layered double hydroxide, Colloids Surf[END_REF][START_REF] Ryu | Layered double hydroxide as novel antibacterial drug delivery system[END_REF]. This would thus reduce the bioavailability of these necessary nutrients and inhibit the bacterial growth. Although these investigations suggested that the rough surface of LDHs aid in the adhesion phenomena leading to the achieved antibacterial effect [START_REF] Zhang | Colloidal Surface Engineering: Growth of Layered Double Hydroxides with Intrinsic Oxidase-Mimicking Activities to Fight Against Bacterial Infection in Wound Healing[END_REF][START_REF] Wang | Flower-like Surface of Three-Metal-Component Layered Double Hydroxide Composites for Improved Antibacterial Activity of Lysozyme[END_REF], real measurements of LDH surface roughness are lacking. One good method to ensure the effectiveness of LDHs roughness in the antibacterial activity is to compare the antibacterial activity of different LDH samples having different surface roughness which could be evaluated easily by atomic force microscopy (AFM)

for example [START_REF] Yu | MgAl-Layered-Double-Hydroxide/Sepiolite Composite Membrane for High-Performance Water Treatment Based on Layer-by-Layer Hierarchical Architectures[END_REF][START_REF] Kim | Surface roughness effect on the cellular uptake of layered double hydroxide nanoparticles[END_REF].

2.3.Antibacterial test assays used to evaluate the direct contact antibacterial performance of LDHs

Different antibacterial test assays allowed the evaluation antibacterial performance of LDHs associated with direct contact. One very important point to consider while correlating the antibacterial performance of LDHs to their followed antibacterial mechanism is the used antibacterial test assay. Generally, evaluating the antibacterial performance of pristine and NPsloaded LDHs could be achieved by agar disk or agar well diffusion methods [START_REF] Moaty | Preparation, characterization and antimicrobial applications of Zn-Fe LDH against MRSA[END_REF][START_REF] Carja | Nanosized silver-anionic clay matrix as nanostructured ensembles with antimicrobial activity[END_REF][START_REF] Dutta | Zn 2 Al-CO 3 Layered Double Hydroxide: Adsorption, Cytotoxicity and Antibacterial Performances[END_REF][START_REF] Awassa | Divalent metal release and antimicrobial effects of layered double hydroxides[END_REF][START_REF] Mishra | Antibacterial actions of silver nanoparticles incorporated Zn-Al layered double hydroxide and its spinel[END_REF],

plate colony count method [49,[START_REF] Qiao | Methyl blue adsorption properties and bacteriostatic activities of Mg-Al layer oxides via a facile preparation method[END_REF], or else by evaluating the minimum inhibitory concentration (MIC) of LDHs by broth macro or microdilution assays (Table 1) [START_REF] Carja | Nanosized silver-anionic clay matrix as nanostructured ensembles with antimicrobial activity[END_REF][START_REF] Dutta | Zn 2 Al-CO 3 Layered Double Hydroxide: Adsorption, Cytotoxicity and Antibacterial Performances[END_REF][START_REF] Dutta | Zn 2 Al-CO 3 Layered Double Hydroxide: Adsorption, Cytotoxicity and Antibacterial Performances[END_REF][START_REF] Awassa | Divalent metal release and antimicrobial effects of layered double hydroxides[END_REF][START_REF] Lobo-Sánchez | ZnAl layered double hydroxides impregnated with eucalyptus oil as efficient hybrid materials against multi-resistant bacteria[END_REF].

However, not all assays are suitable to suggest or validate a certain antibacterial mechanism. For instance, Carja et al. [START_REF] Carja | Nanosized silver-anionic clay matrix as nanostructured ensembles with antimicrobial activity[END_REF] and Mishra et al. [START_REF] Mishra | Antibacterial actions of silver nanoparticles incorporated Zn-Al layered double hydroxide and its spinel[END_REF] suggested direct surface interaction as a mechanism explaining the antibacterial activities of Ag-ZnAl LDH particles. However, they reported inhibitions diameters between 7 and 18 mm in agar-agar gels, which seems in contradiction with a mechanism only driven by contact with the bacteria. Indeed, the mass transfer of particles through convection can be neglected in a gel, therefore the distance run by a particle in those experiments can be estimated with the Fick equation using the Stokes-Einstein equation for the diffusion coefficients [START_REF] Drugeon | Assessment of formulas for calculating critical concentration by the agar diffusion method[END_REF]. At room temperature, in an agar-agar gel (typical viscosity: η = 0.01 Pa.s or 10 cPl) and with standard LDH particle aggregate (5 µm), the diffusion constant is in the order of magnitude of 4-16 m 2 /s. During a standard duration of an experiment of disk diffusion or agar well diffusion (24 to 48h), the typical distance of the particle from the original position is 20 to 40 µm. Direct contact cannot be the explanation for the inhibition of the bacterial growth several millimeters away from the place in which LDHs were deposited. Therefore, inhibition zone diameters measurements are not suitable to evaluate the antibacterial effect of LDHs by direct surface interactions.

2.4.Experimental observations, limitation and maximization of the direct contact effect of LDHs

Haffner et al. investigated the influence of particle size (from 42 to 208 nm) on pristine LDH interactions with bacteria-mimicking membranes (DOPE/DPG) [START_REF] Häffner | Membrane interactions and antimicrobial effects of inorganic nanoparticles[END_REF]. LDH binding to bacteriamimicking membranes and their destabilization increased significantly with decreasing particle size. Due to strong interactions with anionic lipopolysaccharide and peptidoglycan layers, direct membrane disruption was observed for both Gram-negative E. coli and Gram-positive S. aureus bacteria. Such membrane disruption was primarily evidenced by the leakage of the tested membrane liposomes which was enhanced with decreasing LDH particle sizes. The net negative charge of bacteria was also reduced due to the presence of electrostatic surface interactions which were thought to induce the observed membrane disruption.

In fact, the influence of LDH particle sizes on their antibacterial properties could also be [START_REF] Mao | Mussel-inspired nano-silver loaded layered double hydroxides embedded into a biodegradable polymer matrix for enhanced mechanical and gas barrier properties[END_REF]. Fig. 4A shows the transmission electron microscopy (TEM) images of the loaded AgNPs in each composite, the size of loaded Ag-NPs was 25, 65 and 15 nm for Ag-LDHs@TA-Fe(III), Ag-LDHs(PVP) and Ag-LDHs@PDA, respectively. Moreover, the amount of AgNPs loading was determined using energy dispersive spectroscopy (EDS) surface analysis (Fig. 4B).

determined
Consequently, the antimicrobial performance of the Ag-LDHs hybrids was influenced by both the size and amount of loaded Ag NPs. Ag-LDHs(PVP) hybrid, having the highest Ag loading of 3.37% and medium-sized AgNPs, presented the lowest MIC value (Fig. 4C), thus being the best tested antimicrobial composite. On the other hand, even with the lowest Ag loading, Ag-LDHs@TA-Fe(III) showed better antibacterial performance than that of Ag-LDHs@PDA, due to its very small particle sizes of loaded AgNPs. A similar finding was reported by Nocchetti et al. [START_REF] Nocchetti | AgCl-ZnAl Layered Double Hydroxides as Catalysts with Enhanced Photodegradation and Antibacterial Activities[END_REF], where AgCl-LDH composites with Ag loading of 4.9% and small AgCl NPs (64 nm)

showed greater antibacterial performance compared to that of another AgCl-LDH composite with Ag loading of 12.1% but with a larger size (94.8 nm). Therefore, the antibacterial performance was rather dependent on the dimensions of loaded NPs, which corresponded to greater surface area and thereby promoted better contact with the bacteria probably leading to enhanced mobility of NPs adhering to bacteria. In fact, NPs as AgNPs can anchor to the bacterial cell wall and consequently impose membrane damages [START_REF] Khalandi | A Review on Potential Role of Silver Nanoparticles and Possible Mechanisms of their Actions on Bacteria[END_REF][START_REF] Qing | Potential antibacterial mechanism of silver nanoparticles and the optimization of orthopedic implants by advanced modification technologies[END_REF]. After adhesion to the bacterial wall, NPs can also penetrate the membrane to interact with cellular structures and biomolecules such as proteins, lipids, and DNA. Interaction between metallic NPs and cellular structures or biomolecules may subsequently lead to bacterial dysfunction and finally growth inhibition or death [START_REF] Khalandi | A Review on Potential Role of Silver Nanoparticles and Possible Mechanisms of their Actions on Bacteria[END_REF][START_REF] Qing | Potential antibacterial mechanism of silver nanoparticles and the optimization of orthopedic implants by advanced modification technologies[END_REF][START_REF] Godoy-Gallardo | Antibacterial approaches in tissue engineering using metal ions and nanoparticles: From mechanisms to applications[END_REF][START_REF] Sánchez-López | Metal-Based Nanoparticles as Antimicrobial Agents: An Overview[END_REF] The previously mentioned investigations succeeded to correlate direct surface interactions between bacteria and LDHs to their antibacterial activity. However, the used experimental approaches were only achieved through the evaluation of the effect of LDH particle sizes [START_REF] Häffner | Membrane interactions and antimicrobial effects of inorganic nanoparticles[END_REF][START_REF] Mao | Mussel-inspired nano-silver loaded layered double hydroxides embedded into a biodegradable polymer matrix for enhanced mechanical and gas barrier properties[END_REF] or else through qualitative adhesion observations using SEM or TEM imaging [START_REF] Zhang | Colloidal Surface Engineering: Growth of Layered Double Hydroxides with Intrinsic Oxidase-Mimicking Activities to Fight Against Bacterial Infection in Wound Healing[END_REF][START_REF] Wang | Flower-like Surface of Three-Metal-Component Layered Double Hydroxide Composites for Improved Antibacterial Activity of Lysozyme[END_REF] without any quantification of the driving forces of interactions. In fact, other methods can be used to investigate and quantify LDHs-bacterial membrane. In this context, the use of recent AFM-based force spectroscopy modes would be helpful, e.g. single-molecule and single-cell force spectroscopies (SPFS and SCFS, respectively [START_REF] Beaussart | Microbial adhesion and ultrastructure from the singlemolecule to the single-cell levels by Atomic Force Microscopy[END_REF][START_REF] Beaussart | The microbial adhesive arsenal deciphered by atomic force microscopy[END_REF]. The use of such force spectroscopy approaches would allow not only a qualitative but also a quantitative evaluation of the presented forces of interactions at the singleparticle or single-cell levels.

Even though there is still much missing information concerning the role of direct surface interactions in the antibacterial effect of pristine and NPs-loaded LDHs, one could still derive some factors which could help maximize such an effect:

-

LDH antibacterial activity by dissolution

3.1.Release of antibacterial molecules out of antibacterial molecules loaded-LDHs

Most investigations highlighting the efficiency of LDHs to act as antibacterial materials were related to their ability to intercalate antibacterial organic biomolecules such as antibiotics [START_REF] Li | High and long-term antibacterial activity against Escherichia coli via synergy between the antibiotic penicillin G and its carrier ZnAl layered double hydroxide, Colloids Surf[END_REF][START_REF] Ryu | Layered double hydroxide as novel antibacterial drug delivery system[END_REF][START_REF] Li | Phenoxymethylpenicillin-intercalated hydrotalcite as a bacteria inhibitor[END_REF], lysozymes [START_REF] Wang | Flower-like Surface of Three-Metal-Component Layered Double Hydroxide Composites for Improved Antibacterial Activity of Lysozyme[END_REF][START_REF] Yang | Preparation and antibacterial activity of lysozyme and layered double hydroxide nanocomposites[END_REF] and antibacterial polymeric organic molecules [START_REF] Kamal | Bimodal applications of LDH-chitosan nanocomposite: water treatment and antimicrobial activity[END_REF]. This is probably due to LDHs' remarkable property of intercalating a wide variety of anions within their interlamellar regions. Recently, antibacterial molecules-intercalated LDHs had been also incorporated with several polymeric matrices to design efficient antibacterial fillers for various applications as well [START_REF] Behzadi Nia | Carboxymethylcellulose/layered double hydroxides bio-nanocomposite hydrogel: A controlled amoxicillin nanocarrier for colonic bacterial infections treatment[END_REF][START_REF] Coiai | Antibacterial LDPE-based nanocomposites with salicylic and rosmarinic acidmodified layered double hydroxides[END_REF]. Table 2 reports some of the previous work done on the efficiency of antibacterial compounds-loaded LDH composites.

280

Nearly all of the studies dealing with the antibacterial activity of antibacterial organic molecules-281 loaded LDHs are mainly concerned with the controlled release of the active biomolecule out of the 282 LDH matrix [START_REF] Khan | Controlled release of organic–inorganic nanohybrid:cefadroxil intercalated Zn–Al-layered double hydroxide[END_REF][START_REF] Djebbi | Delivery system for berberine chloride based on the nanocarrier ZnAllayered double hydroxide: Physicochemical characterization, release behavior and evaluation of anti-bacterial potential[END_REF][START_REF] Behzadi Nia | Carboxymethylcellulose/layered double hydroxides bio-nanocomposite hydrogel: A controlled amoxicillin nanocarrier for colonic bacterial infections treatment[END_REF][START_REF] Santana-Cruz | Antibacterial Activity of Pipemidic Acid ions-MgFeAl Layered Double Hydroxide Hybrid Against E. coli and S. typhi[END_REF][START_REF] Wang | Intercalation assembly of kojic acid into Zn-Ti layered double hydroxide with antibacterial and whitening performances[END_REF][START_REF] Imran | Dissolution kinetics of pyroaurite-type layered double hydroxide doped with Zn: Perspectives for pH controlled micronutrient release[END_REF][START_REF] Raval | Controlled-release and antibacterial studies of doxycycline-loaded poly(ε-caprolactone) microspheres[END_REF][START_REF] Wang | Controlled drug release characteristics and enhanced antibacterial effect of graphene oxide-drug intercalated layered double hydroxide hybrid films[END_REF], which is the main contributor to the followed antibacterial 283 mechanism. . One example of controlled release behavior of antibacterial molecule-loaded LDHs 284 is the controlled delivery of Kojic acid (KA) out of ZnTi LDH [START_REF] Wang | Intercalation assembly of kojic acid into Zn-Ti layered double hydroxide with antibacterial and whitening performances[END_REF]. In phosphate-buffered saline 285 (PBS, pH = 5), pure KA is released in one step and not gradually. However, the ZnTi-KA 286 composite released KA anions in a slow gradual manner, where even after 12 h KA anions were 287 still released. The prolonged release behavior of KA suggests the maintenance of a long-term 288 antibacterial effect of the LDH hybrid. The long-term antibacterial activity of antimicrobial 289 molecules loaded-LDHs can be explained by two main reasons: (1) the protection of the LDH 290 matrix for loaded molecules by isolating them from a hostile environment, (2) the slow release of 291 loaded molecules from the LDH interlayer [START_REF] Li | High and long-term antibacterial activity against Escherichia coli via synergy between the antibiotic penicillin G and its carrier ZnAl layered double hydroxide, Colloids Surf[END_REF][START_REF] Djebbi | Delivery system for berberine chloride based on the nanocarrier ZnAllayered double hydroxide: Physicochemical characterization, release behavior and evaluation of anti-bacterial potential[END_REF][START_REF] Rives | Intercalation of drugs in layered double hydroxides and their controlled release: A review[END_REF]. Moreover, the intercalation of KA into LDH 292 also improved its stability at higher temperatures. Such enhancement of thermal stability of 293 antibacterial molecules when being loaded in LDH matrices was also reported in other studies as 294 well [START_REF] Kutlu | Preparation of meltspun antimicrobially modified LDH/polyolefin nanocomposite fibers[END_REF][START_REF] Geng | Preparation, characterization, structure-property relationships, and thermal degradation kinetics of poly (lactic acid)/amidated potassium hydrogen phthalate intercalated layered double hydroxides nanocomposites[END_REF][START_REF] Madhusha | A Novel Green Approach to Synthesize Curcuminoid-Layered Double Hydroxide Nanohybrids: Adroit Biomaterials for Future Antimicrobial Applications[END_REF]] 295

Methods for intercalating antibacterial molecules in the LDH and their influence 296 on the release behavior 297

One important factor influencing the antimicrobial activity of antibacterial molecules-LDH hybrids is the applied synthesis method [START_REF] Djebbi | Preparation and optimization of a drug delivery system based on berberine chloride-immobilized MgAl hydrotalcite[END_REF][START_REF] Mishra | Comparative Evaluation of Synthetic Routes and Antibacterial/Antifungal Properties of Zn-Al Layered Double Hydroxides Containing Benzoate Anion[END_REF]. As with any other LDH material, antibacterial molecules loaded-LDHs were prepared by three different methods illustrated in Fig. 5(A): [START_REF] Fitzpatrick | Modelling microbial infection to address global health challenges[END_REF] anion-exchange reactions, (2) direct coprecipitation, (3) rehydration of double layered oxides. Release profiles of BBC from anion exchange and coprecipitation biohybrid LDHs. (C): Inhibitory diameters of free and immobilized BBC. Reproduced from Ref. [START_REF] Djebbi | Preparation and optimization of a drug delivery system based on berberine chloride-immobilized MgAl hydrotalcite[END_REF] with permission from Elsevier.

Copyright (2016), Elsevier.

(1) Anion exchange reactions: due to their remarkable property in terms of anion exchange capacity, antibacterial-molecules LDHs can be synthesized by an indirect anion exchange route, which was commonly used for intercalation of larger anions as biological ones for example [START_REF] Costantino | Ion exchange and intercalation properties of layered double hydroxides towards halide anions[END_REF].

In this method LDHs with host anions of NO3 -, CO3 2-, and Cl -are primarily prepared by coprecipitation, then the exchange with other desired intercalated anions can take place (Fig. 5A) [START_REF] Rives | Intercalation of drugs in layered double hydroxides and their controlled release: A review[END_REF][START_REF] Benício | LAYERED DOUBLE HYDROXIDES: NANOMATERIALS FOR APPLICATIONS IN AGRICULTURE[END_REF]. In fact, anion-exchange synthesis is the most frequently used method to synthesize antibacterial molecules-intercalated LDHs [START_REF] Mishra | Orientation of Organic Anions in Zn-Al Layered Double Hydroxides with Enhanced Antibacterial Property[END_REF][START_REF] Wang | Synthesis, characterization, and controlled release antibacterial behavior of antibiotic intercalated Mg-Al layered double hydroxides[END_REF][START_REF] Ryu | Layered double hydroxide as novel antibacterial drug delivery system[END_REF][START_REF] Kutlu | Preparation of meltspun antimicrobially modified LDH/polyolefin nanocomposite fibers[END_REF][START_REF] Costantino | New Polymeric Composites Based on Poly(ϵ-caprolactone) and Layered Double Hydroxides Containing Antimicrobial Species[END_REF].

(2) Coprecipitation: coprecipitation takes place by the addition of a solution containing the anionic form of the desired antibacterial molecule to be intercalated into another solution having the divalent and trivalent metal cations in a specific ratio. The pH should further be increased by simultaneous addition of a base usually sodium hydroxide (NaOH) or urea hydrolysis that subsequently leads to the precipitation of the antibacterial molecule-intercalated LDH (Fig. 5A) [START_REF] Djebbi | Delivery system for berberine chloride based on the nanocarrier ZnAllayered double hydroxide: Physicochemical characterization, release behavior and evaluation of anti-bacterial potential[END_REF][START_REF] Li | Phenoxymethylpenicillin-intercalated hydrotalcite as a bacteria inhibitor[END_REF][START_REF] Bugatti | Influence of the powder dimensions on the antimicrobial properties of modified layered double hydroxide[END_REF]. Although the direct coprecipitation method does not favor the intercalation of large molecules inside the interlamellar space of the LDH [START_REF] Das | Heteropoly acid intercalated Zn/Al HTlc as efficient catalyst for esterification of acetic acid using n-butanol[END_REF], several investigations on the synthesis of antibacterial molecules-intercalated LDHs using the coprecipitation method were reported [START_REF] Djebbi | Delivery system for berberine chloride based on the nanocarrier ZnAllayered double hydroxide: Physicochemical characterization, release behavior and evaluation of anti-bacterial potential[END_REF][START_REF] Bugatti | Influence of the powder dimensions on the antimicrobial properties of modified layered double hydroxide[END_REF].

(3) Rehydration of double layer oxides: one property of LDH clay materials is their ability to regain their original structure back after calcination followed by rehydration, this property is termed as "memory effect" [START_REF] Rocha | Reconstruction of layered double hydroxides from calcined precursors: a powder XRD and 27Al MAS NMR study[END_REF]. Calcining LDHs at mild temperatures (400-500ºC) would transfer them to their respective layered double oxides by eliminating interlayer water molecules, hydroxyl groups, and previously intercalated anions. Furthermore, when these obtained metal oxides are immersed in another solution containing other anions to be intercalated (antibacterial anions in this case), the LDH structure can be regenerated back by rehydration (Fig. 5A) [START_REF] Erickson | A study of structural memory effects in synthetic hydrotalcites using environmental SEM[END_REF].

Several antibacterial molecules were successfully loaded into the LDH interlamellar spacing using this synthesis method [START_REF] Li | Phenoxymethylpenicillin-intercalated hydrotalcite as a bacteria inhibitor[END_REF][START_REF] Wang | Synthesis, sustained release properties of magnetically functionalized organic-inorganic materials: Amoxicillin anions intercalated magnetic layered double hydroxides via calcined precursors at room temperature[END_REF].

Djebbi et al. compared the antibacterial behavior of the same LDH composite synthesized using two different methods, conventional anion exchange (AE) and direct coprecipitation (AC) routes [START_REF] Djebbi | Preparation and optimization of a drug delivery system based on berberine chloride-immobilized MgAl hydrotalcite[END_REF]. The amount of loaded antibacterial molecule berberine chloride (BBC) in the inorganic matrix, estimated by UV-Vis spectroscopy analysis, was found to be 24% and 93% in BMAE and BMAC, respectively. Moreover, the release profiles of BBC from AE and AC biohybrid LDHs were separately measured in PBS at pH = 7.4 (Fig. 5B), and the released percentage of BBC from AC was greater than that from AE. Consequently, the antimicrobial activity of AC was more efficient than that of AE (Fig. 5C) due to an improvement in chemical stability, the greater amount of loaded and released BBC. On contrary, Mishra et al. pointed out the lower antimicrobial efficiency and lower release behavior of Benzoate-LDH hybrids synthesized by coprecipitation in comparison to those synthesized by anion-exchange and reconstruction methods [START_REF] Mishra | Comparative Evaluation of Synthetic Routes and Antibacterial/Antifungal Properties of Zn-Al Layered Double Hydroxides Containing Benzoate Anion[END_REF]. The authors attributed the difference in the antimicrobial performance of the hybrids synthesized using different methods to the different orientations of benzoate anions within the interlamellar space of the LDH in each case. Such different orientation resulted in a different loading and release behavior of antibacterial molecules as previously suggested by Wang et al. [START_REF] Wang | Synthesis, characterization, and controlled release antibacterial behavior of antibiotic intercalated Mg-Al layered double hydroxides[END_REF]. Such contradiction suggests that choosing a particular synthesis method to synthesize antibacterial molecules-loaded LDHs is an important factor to be taken into consideration. Therefore, an optimization of the most efficient synthesis method should be performed according to the type, structure, and orientations of the antibacterial molecules loaded in the LDH interlayer.

Influence of the LDH host on the antibacterial activity of antibacterial moleculesloaded LDH composites

Bugatti et al. reported the influence of powder dimensions evaluated by granulometry analysis on the antimicrobial properties of antibacterial molecules-loaded LDHs [START_REF] Bugatti | Influence of the powder dimensions on the antimicrobial properties of modified layered double hydroxide[END_REF]. Antibacterial composites (ZnAl-o-BzOH) based on ZnAl LDH and o-hydroxybenzoate (o-BzOH), also known as salicylate, were synthesized by a direct coprecipitation method. The initial powder dimensions were reduced by the high energy ball milling (HEBM) technique for 1 to 5 min influencing the size as well as the morphology of the powders. Large, agglomerated particles were found for the pristine samples, while well-defined platelets with uniform and thinner sizes were present in the milled samples. The antimicrobial activity against E. coli was found much more effective for the samples with lower powder dimensions. Therefore, particles with lower dimensions provided an enhanced release of o-BzOH anions and an enhanced antibacterial activity. A similar finding was suggested by Kuznetsova et al. who reported a remarkable impact of the hydrodynamic radius of the LDH composite powders on the release behavior of 2-mercaptobenzothiazole anions out of ZnAl LDH host and therefore an enhanced antibacterial activity [START_REF] Kuznetsova | Antimicrobial activity of 2-mercaptobenzothiazole released from environmentally friendly nanostructured layered double hydroxides[END_REF].

Moreover, Coiai et al. had recently reported the contribution of the host LDH to the antimicrobial activity of antibacterial molecules-modified LDHs [START_REF] Coiai | Antibacterial LDPE-based nanocomposites with salicylic and rosmarinic acidmodified layered double hydroxides[END_REF], where salicylic acid SA-ZnAl LDH hybrids provided higher antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus bacteria in comparison to SA-MgAl LDH hybrids. The authors attributed the enhanced antibacterial activity of SA-ZnAl to the presence of zinc ions in the structure which may have contributed to the antibacterial performance of the hybrid alongside the antibacterial action of SA.

The specific effect of Zn 2+ ions as an antimicrobial agent will be detailed in section 3.2.1. Such synergistic antibacterial effect of Zn 2+ ions released out ZnAl LDHs and organic molecules loaded in the LDH matrix was also reported by Li et al. [START_REF] Li | High and long-term antibacterial activity against Escherichia coli via synergy between the antibiotic penicillin G and its carrier ZnAl layered double hydroxide, Colloids Surf[END_REF].

Principles for the design of an efficient antibacterial molecules-loaded LDH

When designing antibacterial molecules-loaded LDH composites of controlled release behavior, several factors must be taken into consideration to provide an enhanced antibacterial behavior including:

-the efficiency of the antibacterial guest molecule itself -the synthesis method of the composite and the orientation of antibacterial molecule within the interlamellar spacing of the LDH matrix allowing maximum loading -the size and dimensions of the resulted composite controlling the release behavior of the hosted antibacterial biomolecule -the type of LDH host matrix aiding in the antibacterial performance

Dissolution of metallic cations out of pristine and NPs-loaded LDHs

As suggested previously, the release of metallic ions is one other possible mechanism explaining the antibacterial activity of pristine and NPs-loaded LDHs (Table 1). Metallic ions as Zn 2+ , Cu 2+ and Ag + can damage bacterial membrane functions by: (i) blocking the transport of nutrients and substances in and out of the membrane, (ii) reducing protein and enzymatic expressions, (iii) or else by binding to bacterial intracellular DNA leading to their denaturation [START_REF] Peng | The prospect of layered double hydroxide as bone implants: A study of mechanical properties, cytocompatibility and antibacterial activity[END_REF][START_REF] Borkow | Copper as a Biocidal Tool[END_REF][START_REF] Mittapally | Metal ions as antibacterial agents[END_REF][START_REF] Ishida | Antibacterial mechanism of Ag+ ions for bacteriolyses of bacterial cell walls via peptidoglycan autolysins, and DNA damages[END_REF][START_REF] Midori-Ku | Comparative Bacteriolytic Mechanism for Ag+ and Zn2+ Ions against Staphylococcus Aureus and Escherichia Coli: A review[END_REF].

Antimicrobial effect through the release of Zn 2+ , Cu 2+ and Ag +

Moaty et al. reported an efficient antibacterial activity against several Gram-positive and Gramnegative bacterial strains for ZnFe pristine LDH which was slightly weaker than that imposed by standard antibiotics [START_REF] Moaty | Preparation, characterization and antimicrobial applications of Zn-Fe LDH against MRSA[END_REF]. The authors suggested that released zinc ions had a great effect on both bacteria because of the binding to their membranes which prolonged the lag phase of the growth cycle. Dutta et al. also reported a significant impact of Zn2Al-CO3 LDH on several Gram-positive and Gram-negative bacteria. The antibacterial efficiency varied in accordance with the applied dosage of Zn2Al-CO3 LDH, where it was greatly enhanced for growing concentrations.

Additionally, the antibacterial activity was also dependent on the type of tested bacteria. Among the tested microorganisms, Gram-negative bacteria were comparatively more sensitive toward the LDH sample than Gram-positive ones. The authors reported that released Zn 2+ could strongly contribute to the observed antibacterial effect. Finally, the difference in the antibacterial activity between Gram-negative and Gram-positive bacteria was clarified by the different structures and the chemical composition of the cell surfaces of these two types of bacteria. Gram-positive bacteria possess an extra outer membrane that covers the peptidoglycan layer that is not present in Gramnegative bacteria [START_REF] Amornpitoksuk | Synthesis, photocatalytic and antibacterial activities of ZnO particles modified by diblock copolymer[END_REF].

Vallejo et al. reported the influence of the nature and amount of constituent divalent metal on the antibacterial activity of pristine LDHs [START_REF] León-Vallejo | Study of layered double hydroxides as bactericidal materials against Corynebacterium ammoniagenes, a bacterium responsible for producing bad odors from human urine and skin infections[END_REF]. LDH samples containing copper or magnesium as divalent metals were found to be less active against Corynebacterium ammoniagenes, whereas those containing zinc showed superior bactericidal activity ranging from 98.1% to 99.9% depending on the applied dosage. The reason behind the enhanced activity of Zn-based LDHs was linked to the superior efficiency of the released zinc ions to induce an antibacterial effect than other released ions as Cu 2+ and Mg 2+ . The impact of the nature of constituent divalent metal was also reported by Li et al. who studied the antibacterial activity of different MAl-LDH (M: Mg 2+ , Cu 2+ , Ni 2+ , Co 2+ , and Mn 2+ ) against Gram-negative E. coli, and S. aureus Gram-positive bacteria.

When the concentration of MAl-LDHs increased up to 1500 µg/mL, CuAl-LDH and MnAl-LDH showed the best bacterial inhibition compared to the other LDH samples. On the other hand, NiAl-LDH and CoAl-LDH exhibited a limited antibacterial activity, whereas MgAl-LDH had no antibacterial ability. Similarly, Peng et al. compared the antimicrobial activity of MgAl and MgFe LDHs to those of ZnAl and ZnFe LDHs, where the two latter presented superior antibacterial properties [START_REF] Peng | The prospect of layered double hydroxide as bone implants: A study of mechanical properties, cytocompatibility and antibacterial activity[END_REF]. A global investigation of the impact of different released constituent divalent metallic cations of MAl-LDHs (M: Mg 2+ , Cu 2+ , Ni 2+ , Co 2+ , and Zn 2+ ) was also performed recently by our group [START_REF] Awassa | Divalent metal release and antimicrobial effects of layered double hydroxides[END_REF], where it was found out that only Cu and Zn-based LDHs releasing Cu 2+ and Zn 2+ ions were able to impose an efficient antibacterial effect against S. aureus and E. coli bacteria.

The inhibition zone diameters of all LDH samples were very similar to those of their corresponding metallic salts counterparts (having a concentration similar to that of the one released by LDHs, thus suggesting the strong relation between the antibacterial activity of pristine LDHs and their metallic ions release capacity. Additionally, we figured out that not only the nature of constituent divalent metals could impact the antibacterial effect of pristine LDH but also the stability and crystallinity of LDHs. For instance, exchanging carbonate anions with other anions having lower intercalating affinities as Cl -, NO3 -and ClO4 -, increasing the Zn(II):Al(III) molar ratio [START_REF] Awassa | Divalent metal release and antimicrobial effects of layered double hydroxides[END_REF][START_REF] Lobo-Sánchez | ZnAl layered double hydroxides impregnated with eucalyptus oil as efficient hybrid materials against multi-resistant bacteria[END_REF] and lowering the crystallinity enhanced the release of Zn 2+ in aqueous broth media and subsequently the showed greater inhibition zone diameters and lower MIC values.

Tabti et al. synthesized a range of Cu-LDHs having different molar ratios of Cu:Al (Cu0.05-Al0.15, Cu0.10-Al0.10, Cu0.14-Al0.06, and Cu0.15-Al0.05) [START_REF] Tabti | Facile synthesis of Cu-LDH with different Cu/Al molar ratios: application as antibacterial inhibitors[END_REF]. The antibacterial activity of the synthesized samples was estimated toward several bacterial strains, e.g. E. coli, P. aeruginosa, E. faecalis, S. aureus, and B. Subtilis. Samples having a higher molar ratio of Cu:Al showed better antibacterial activity due to the presence of a greater amount of Cu 2+ ions which were further ejected providing an enhanced impact.

Huang et al. reported the fabrication of hybrid polypyrrole-silver coated layered double hydroxides (LDHs@PPy-Ag) for both enhanced gas carrier property and antibacterial activity [START_REF] Huang | Multifunctional Polypyrrole-silver Coated Layered Double Hydroxides Embedded into a Biodegradable Polymer Matrix for Enhanced Antibacterial and Gas Barrier Properties[END_REF]. The prepared LDH hybrid was able to completely inhibit the growth of bacteria. The antibacterial activity of the prepared LDH hybrid was attributed to the synergistic effect of polypyrrole and the loaded silver NPs. In particular, it was believed that the direct contact effect with loaded AgNPs alongside the Ag + ions released from unstable AgNPs were responsible for the excellent observed antibacterial activity.

Yadollahi et al. prepared antibacterial nanocomposite hydrogels by the combination of carboxymethyl cellulose (CMC), LDH and silver nanoparticles (AgNPs) [START_REF] Yadollahi | Antibacterial carboxymethyl cellulose/Ag nanocomposite hydrogels cross-linked with layered double hydroxides[END_REF]. CMC-LDH hydrogels were prepared by intercalating CMC into different LDHs, e.g. MgAl, NiAl, CuAl and ZnAl LDHs. Ag/CMC-LDH nanocomposite hydrogels (Ag/CMC-Mg-LDH, Ag/CMC-Ni-LDH, Ag/CMC-Cu-LDH and Ag/CMC-Zn-LDH) were prepared through in situ formation of AgNPs within the CMC-LDHs. CMC-Mg-LDH and CMC-Ni-LDH showed no antibacterial activity against E. coli and S. aureus. On contrary, an antibacterial effect was observed for both CMC-Cu-LDH and CMC-Zn-LDH nanocomposites. Such an effect was attributed to the antibacterial features of Cu 2+ and Zn 2+ ions released from CMC-Cu-LDH and CMC-Zn-LDH nanocomposites, respectively. Ag/CMC-LDH nanohybrids exhibited a superior antibacterial activity against both bacteria in comparison to their CMC-LDH counterparts. The authors attributed such enhanced antibacterial activity to the antibacterial effect of released Ag + ions from Ag/CMC-LDH nanohybrids. Metal ions were slowly released from metallic NPs as AgNPs, and then absorbed through the cell membrane. The released silver metallic ions were probably able to interact with functional groups of nucleic acids and proteins, e.g. mercapto (-SH), amino (-NH), carboxyl (-COOH) groups [START_REF] Wang | The antimicrobial activity of nanoparticles: present situation and prospects for the future[END_REF]. This could further affect the normal physiological processes by damaging the enzymatic activity, changing the cell structure, and ultimately inhibiting the growth of microorganisms [START_REF] Sánchez-López | Metal-Based Nanoparticles as Antimicrobial Agents: An Overview[END_REF][START_REF] Wang | The antimicrobial activity of nanoparticles: present situation and prospects for the future[END_REF].

Correlation to the amount of metallic ions released and principles for the design of LDHs with optimal antibacterial effect through ion release

Although all the previously mentioned investigations suggest the contribution of released metallic ions to the antibacterial effect of pristine and NPs-loaded LDHs, the literature lacks real experimental setups validating such contribution, e.g. quantitative evaluation of the concentration of released ions in solution and their antibacterial efficiency. In our study [START_REF] Chaillot | Layered double hydroxides and LDH-derived materials in chosen environmental applications: a review[END_REF], the MIC values of Zn-based LDHs were studied in accordance with the amount of zinc ions released in LB and TSB broth media. The MIC values of Zn-based LDHs decreased significantly from 12 to 0.375 mg/mL when higher amounts of zinc ions were released. Therefore, we were able to establish the contribution of metallic ions released out of pristine LDHs to their antibacterial properties thus confirming the suggested antibacterial mechanism of metallic ions release.

In summary, several properties of pristine and NPs-loaded LDHs can be tuned in a way that the antibacterial effect by metallic ions release could be enhanced, including:

-nature of constituent divalent metal or loaded NP to be released, e.g. Zn 2+ , Cu 2+ and Ag + , having the greater effect -increasing the release of the divalent cations in various ways:

LDH antibacterial activity by the generation of reactive oxygen species

Reactive oxygen species (ROS) are produced by bacteria itself (endogenous) as a byproduct of aerobic respiration or generated in the host environment (exogenous) [START_REF] Li | Reactive Oxygen Species in Pathogen Clearance: The Killing Mechanisms, the Adaption Response, and the Side Effects[END_REF]. ROS were called "double-edged swords of life" in the mechanism of pathogen clearance [START_REF] Mittler | ROS Are Good[END_REF]. First, successful pathogens can exploit endogenous ROS for their growth and adaption. Second, exogenous ROS were thought to be the weapon providing an efficient antibacterial effect since they can directly damage bacterial membranes, DNA, lipids, and proteins by oxidative stress [START_REF] Li | Reactive Oxygen Species in Pathogen Clearance: The Killing Mechanisms, the Adaption Response, and the Side Effects[END_REF][START_REF] Mittler | ROS Are Good[END_REF][START_REF] Ezraty | Oxidative stress, protein damage and repair in bacteria[END_REF].

Generation of ROS by LDHs

When a suitable source of light irradiates LDHs, an electron (𝑒̅ ) and a hole (ℎ + ) are created which could subsequently generate ROS by the following reactions [START_REF] Abdolmohammad-Zadeh | In situ generation of H2O2 by a layered double hydroxide as a visible light nano-photocatalyst: Application to bisphenol A quantification[END_REF]:

ℎ + + 𝐻₂𝑂 ⟶ 𝐻 + + ⦁𝑂𝐻 (1)
ℎ + + 𝑂𝐻 -⟶ ⦁𝑂𝐻 (2)
𝑒̅ + 𝑂₂ ⟶ ⦁𝑂₂

⦁𝑂₂ + 𝐻⁺ ⟶ ⦁𝐻𝑂𝑂 (4)

⦁𝐻𝑂𝑂 + ⦁𝐻𝑂𝑂 ⟶ 𝐻₂𝑂₂ + 𝑂₂ (5)
Where ⦁OH are hydroxyl radicals, ⦁O₂ are superoxide radical anions, ⦁HOO are hydrogen superoxide radicals ,and H₂O₂ are hydrogen peroxide molecules, all belonging to the family of ROS. The generation of ROS by LDHs is another possible mechanism explaining their antibacterial activity as suggested by several studies reported in Table 1. GNR@LDH-PEG exhibited a significant antibacterial effect in 3 minutes of irradiation and even a higher effect (99.25% and 88.44% for E.coli and S. aureus, respectively). Moreover, the SEM images shown in Fig. 6B and C revealed that both bacteria adhered to GNR@LDH nanostructures, probably due to their positively charged surface. However, before being irradiated by 808 nm laser, no obvious damages were detected on the bacterial membrane of the rod-shaped E. coli and spherical S. aureus (Fig. 6B). On the contrary, under irradiation with a NIR laser for 3 minutes, some crumbles appeared on the surface and the cell membranes became incomplete, distorted, and even melted (Fig. 6C, red arrows). Such observation suggested that bacterial adhesion to LDH nanostructures is not responsible for their antibacterial effect, but it rather served as a first step in promoting the antibacterial activity, as previously suggested by Gottenbos et al. [START_REF] Gottenbos | Antimicrobial effects of positively charged surfaces on adhering Gram-positive and Gram-negative bacteria[END_REF]. In summary, GNR@LDH showed remarkable photothermal antibacterial activity on both Gram-positive and Gram-negative bacteria. This consequently led to the damage and destruction of E. coli and S. aureus bacterial membranes. aureus; *P < 0.01, 0.001 < **P < 0.005, ***P < 0.001, compared with control group, red arrows correspond to distorted cell membranes. Reproduced with permission from Ref. [START_REF] Ma | Core-Shell Gold Nanorod@Layered Double Hydroxide Nanomaterial with Highly Efficient Photothermal Conversion and Its Application in Antibacterial and Tumor Therapy[END_REF]. Copyright (2019), American Chemical Society.

ROS generation by pristine

It is somehow difficult to determine whether the observed damaged bacterial membranes were indeed due to the generation of ROS or the reported photothermal activity under irradiation. To validate such a hypothesis, the morphology of irradiated untreated bacteria should be examined as well.

4.4.Limitation of the attribution of the ROS mechanism and general considerations for its effect

The aforementioned studies suggested either the contribution of ROS [START_REF] Li | Efficient antimicrobial properties of layered double hydroxide assembled with transition metals via a facile preparation method[END_REF][START_REF] Tabti | Facile synthesis of Cu-LDH with different Cu/Al molar ratios: application as antibacterial inhibitors[END_REF]49,[START_REF] Lobo-Sánchez | ZnAl layered double hydroxides impregnated with eucalyptus oil as efficient hybrid materials against multi-resistant bacteria[END_REF][START_REF] Nocchetti | AgCl-ZnAl Layered Double Hydroxides as Catalysts with Enhanced Photodegradation and Antibacterial Activities[END_REF][START_REF] Qiao | Methyl blue adsorption properties and bacteriostatic activities of Mg-Al layer oxides via a facile preparation method[END_REF] or the photothermal effect [START_REF] Ma | Core-Shell Gold Nanorod@Layered Double Hydroxide Nanomaterial with Highly Efficient Photothermal Conversion and Its Application in Antibacterial and Tumor Therapy[END_REF] to the antibacterial mechanism of pristine and NPs-loaded LDHs. A real measurement of ROS and the evaluation of their role is therefore necessary to distinguish the contribution of each to the antibacterial activity. ROS generation could be evaluated using different methods including fluorescence spectroscopy using fluorescent dye probes, ESR and chromatography methods [START_REF] Zhang | Methods for the detection of reactive oxygen species[END_REF].

Another helpful suggestion to ensure the contribution of the generation of ROS by LDHs to their antibacterial activity is to study the antibacterial effect of LDHs in function of generated ROS initially evaluated using both qualitative and quantitative approaches. For example, Dutta et al.

confirmed that the antibacterial effect of ZnO NPs was related to bacterial membrane damage caused by generated ROS evaluated using fluorescent dye probes [START_REF] Dutta | Studies on antibacterial activity of ZnO nanoparticles by ROS induced lipid peroxidation[END_REF]. Additionally, a scavenger of ROS (histidine) was also used in different concentrations during antibacterial experiments in order to evaluate more precisely the role of the generated ROS in the adapted antibacterial mechanism. The antibacterial activity of ZnO NPs decreased significantly with increasing concentrations of added histidine thus ensuring the great contribution of generated ROS to the antibacterial performance of ZnO NPs.

In summary, ROS generation had been evidenced only for pristine LDHs (ZnTi LDH nanosheets).

The possibilities for ROS generation are increased with nanoparticle loading, either with metallic particles (Au), halogen-metal salts (AgCl) or oxides (ZnO).

ROS generation should be triggered by UV, visible or IR light. Of course, a reference measurement must be performed to ensure that the light source itself does not contribute to the observed antibacterial effect.

Conclusion

As summarized in this review, LDH-based materials, e.g., pristine LDHs, NPs loaded-LDHs and antibacterial molecules-loaded LDHs, could be potentially used as efficient antibacterial agents against various Gram-positive and Gram-negative bacterial strains.

(1) Direct surface interactions between negatively charged bacterial membranes and positively charged pristine or NPs-loaded LDHs constitute one possible mechanism explaining their antibacterial activity. Such interaction is maximized by substantial surface charges, smaller particle sizes and good dispersion of LDHs or loaded NPs to promote better interaction with bacterial membranes leading to their damage.

(2) Dissolution of antibacterial molecules from antibacterial molecules-loaded LDH composites is believed to be the main contributor to their antibacterial effect. The induced antibacterial effect by the controlled release of loaded antibacterial molecules is dependent on the efficiency and amount of the loaded antibacterial molecules, their orientation within the LDH matrix, the size of the composites controlling the dissolution behavior as well as the nature of the LDH host which could also aid in the antibacterial effect of these composites. Moreover, dissolution also occurs for pristine and NPs-loaded LDHs that partially release their constituent divalent metallic ions (especially Zn 2+ and Cu 2+ ) and NPs metallic ions (Ag + ), respectively. The release behavior of these metallic ions is dependent on several factors and is increased for lower crystallinity of the particles, weakly interacting intercalated anions, larger divalent over trivalent ion ratio. The released metallic ions can further cause damages to the membrane functions by blocking the transport of nutrients and substances in and out the membrane, or by binding to bacterial intracellular DNA and proteins leading to their denaturation or deactivation.

(3) Finally, upon being radiated by a suitable source of illumination, pristine (Ti-Zn LDH) as well as NPs-loaded (Ag@AgCl, ZnO and Au) LDHs can generate reactive oxygen species (ROS) such as ⦁HO, ⦁O₂ and H₂O₂. These generated ROS are well known to be toxic to bacteria by which they can damage their bacterial membranes, DNA, lipids, and proteins by oxidative stress.

Although these different mechanisms were proposed to explain the antibacterial mode of action of LDH-based materials, experimental evidences verifying them are still lacking. Hence, special investigations are highly encouraged to study in detail the antibacterial mechanisms of LDH-based materials. So that they could potentially be utilized as antibacterial agents in different fields of applications such as antibacterial coatings, food packaging products, cement additives, wound healing and textiles products.

Fig. 1 .

 1 Fig. 1. (A) A schematic representation of LDH structure. The laminar composition is made up of positively

Fig. 2 .

 2 Fig. 2. Mechanism of B. subtilis adhesion onto carbonate intercalated NiFe LDH. (A) Electrostatic

and

  Gram-positive Staphylococcus aureus bacteria, by standard cell viability Live/Dead Bac Light assay (Fig.3Fig. 3A). The ratio of green (alive bacteria) to red (dead bacteria) fluorescence (G/R) was also measured to compare the antibacterial efficiency of the different tested samples. As shown in Fig.3B, the ratio of G/R fluorescence in both Gram-positive and Gram-negative bacteria decreased significantly over time for hollow Mn/Ni LDHs as well as SiO2@Mn/Ni LDHs samples, thus indicating their efficient antibacterial activity. The antibacterial activity of Mn/Ni LDHs and SiO2@Mn/Ni LDHs was related to their bacterial adhesion properties to rough surfaces. As shown in Fig.3C and D, both hollow Mn/Ni LDHs and SiO2 nanorods@Mn/Ni LDHs exhibited relatively good adhesion (blue arrows) and strong connection (red arrows) with E.coli. Therefore, the antibacterial activity of hollow Mn/Ni(OH)x LDHs structures was attributed to their ability to "seize" the bacteria thus disturbing its motility and leading to its death. Such findings come in line with those of Wang et al. who reported the enhancement of antibacterial function by loading lysozyme at the surface of ternary MgZnAl LDHs due to the rough surface of the host flower-like

Fig. 3 .

 3 Fig. 3. (A) Confocal microscopy images of S. aureus and E. coli incubated with a mixture of propidium

Fig. 4 .

 4 Fig. 4. (A) HRTEM images of (a) Ag-LDHs(PVP), (b) Ag-LDHs@PDA and (c) Ag-LDHs@TA-

Fig. 5 .

 5 Fig. 5. (A) Schematic representation of the three different methods used to synthesize antibacterial

o

  increasing the divalent to the trivalent metal molar ratio of LDH (M II :M III ) o lowering the crystallinity of LDH powders o introducing intercalating anions having lower affinity to the layers o decreasing the size of LDHs or loaded NPs which could provide an impact on the amount of metallic ions released, e.g. smaller particles release more metallic ions -tuning the amount and stability of loaded NPs for NPs-loaded LDHs

LDHs 4 . 3 .ROS generation by supported nanoparticles on LDH 4 . 3 . 1 .

 43431 Zhao et al. reported the synthesis of antibacterial ZnTi LDH nanosheets with lateral dimensions in the range of40-80 nm [124]. ESR and X-ray photoelectron spectroscopy (XPS) measurements revealed that Ti 3+ sites were generated within these nanosized LDH platelets. The antibacterial activity of ZnTi LDHs found upon visible-light illumination was explained by the generation of superoxide anion radicals (⦁𝐎₂ ) and hydroxyl radicals by trapping of electrons by Ti 3+ ions and the reaction of generated holes with water or hydroxyl ions(⦁𝐎𝐇), respectively. These generated ROS (⦁𝐎₂ and ⦁𝐎𝐇) were believed to be strong and nonselective oxidants, which could lead to the damage of bacteria by attacking cell membrane to cause cell lysis. Ag@AgCl-generated ROSNocchetti et al. successfully synthesized antibacterial LDH hybrids by loading AgCl NPs on ZnAlLDHs for Rhodamine B (RhB) dye photodegradation and antibacterial potential applications[START_REF] Nocchetti | AgCl-ZnAl Layered Double Hydroxides as Catalysts with Enhanced Photodegradation and Antibacterial Activities[END_REF].Kinetic experiments of the antibacterial activity of the prepared hybrids in the dark and under irradiation (λ > 350 nm). No bacterial growth reduction was observed until 180 min upon treatment with LDH hybrids in dark conditions. On the other hand, the number of live bacteria treated with LDH hybrids was considerably reduced after 60 min of irradiation. The superior antibacterial activity of the nanocomposites under irradiation for a very short time was explained by the formation of Ag clusters on AgCl and the production of reactive oxygen species (ROS), such as ⦁O₂ , which was previously observed in the photodegradation process of RhB in presence of ROS quenchers.

Fig. 6 .

 6 Fig. 6. (A) Antibacterial activity evaluation of GNR@LDH-PEG against: (a) E. coli, (b): S. aureus. (B)

Table 1 .

 1 Antibacterial performance of pristine and NPs-loaded LDHs and the proposed associated antibacterial mechanisms. MIC values correspond to the minimum inhibitory concentration required to inhibit the growth of a certain bacteria, whereas inhibition zone diameters correspond to the obtained inhibitory diameter zone generated by LDHs when being tested in agar disk or agar well diffusion tests.

	LDH sample	Type	Tested Bacteria	Antibacterial performance	Proposed mechanism	Reference

  Zn-based pristine LDHs (micron-sized agglomerated LDHs)[START_REF] Lobo-Sánchez | ZnAl layered double hydroxides impregnated with eucalyptus oil as efficient hybrid materials against multi-resistant bacteria[END_REF], which is remarkably lower than that of ~250 nm-sized Zn-based pristine LDH nanosheets prepared by Dutta et al. (MIC value of 200 μg/mL)[START_REF] Dutta | Zn 2 Al-CO 3 Layered Double Hydroxide: Adsorption, Cytotoxicity and Antibacterial Performances[END_REF]. Moreover, smaller-sized Zn-based LDH nanosheets (~50 nm) investigated by Moaty et al. presented even a lower MIC value of 15.6 μg/mL against the same bacterial strain[START_REF] Moaty | Preparation, characterization and antimicrobial applications of Zn-Fe LDH against MRSA[END_REF]. In this context, LDH nanosheets present a larger surface area and more active sites compared to bulk LDHs. Additionally, the great difference in MIC values between bulk LDHs and LDH nanosheets could also be related to the ability of nanosheets to provide a stable mobile suspension

by comparing the MIC values of LDHs having different particle sizes against the same microorganism. For instance, Lobo-Sanchez et al. reported a MIC value of 10,000 μg/mL for bulk in solution, whereas bulk LDHs tend to sediment, thus limiting the accessible surface and contact with the tested S. aureus bacteria. Mao et al. designed three different Ag loaded-LDHs composites using three different synthesis methods

Table 2 .

 2 Antibacterial performance of organic biomolecules-loaded LDHs reported in the literature. MIC values correspond to the minimum inhibitory concentration required to inhibit the growth of a certain bacteria, whereas inhibition zone diameters correspond to the obtained inhibitory diameter zone generated by antimicrobial molecules-loaded LDHs when being tested in agar disk or agar well diffusion tests.

	LDH host	Antimicrobial compound		Tested Bacteria	Antibacterial performance	Reference
					Penicillin-LDH	showed	efficient
	MgAl	Phenoxymethyl-penicillin Gram-positive: S. aureus	antibacterial activity with inhibition zone diameter ranging from 8 to 25 mm	[84]
					depending on the pH of the media
					Cephazolin-LDH	possessed	an
	ZnAl	Cephazolin		Gram-positive: S. aureus	inhibition zone of 225 m 2 in water and	[47]
					240 m 2 in presence of 0.8% NaCl
					O-BzOH-LDH was able to reduce the
					growth of bacteria which was evidenced
	ZnAl	O-hydroxybenzoate (o-BzOH)	Gram-negative: E. coli	by antimicrobial properties of O-BzOH-plate count method. The	[89]
					LDH were enhanced with decreasing
					powder dimensions.
					LYZ-LDHs	possessed	great
					antibacterial activity (ranging from 40 to
	MgAl	Lysozyme (LYZ)		Gram-positive: S. aureus	97.5%) depending on the LYZ/LDH	[85]
					ratio and the pH of the bacteria-
					containing water
	MgAl	Camphorsulfonic Ciprofloxacin	acid,	Gram-positive: S. aureus, S. epidermidis, S. pyogenes Gram-negative: E. coli, Proteus vulgaris, P. aeruginosa, cloacae Enterobacter	Camphorsulfonic acid-LDH composite was active against Gram-negative negative with higher MIC of 1.3 mg/mL against both Gram-positive and Gram-bacteria with MIC between 1.4 and10 mg/mL, Ciproflaxin-LDH was active	[90]

Acknowledgment

The authors acknowledge the financial support from the French Ministry of Higher Education (MESR), the French National Scientific Centre (CNRS).