Metallo-Glycodendrimeric Materials against Enterotoxigenic Escherichia coli

Conjugation of carbohydrates to nanomaterials has been extensively studied and recognized as an alternative in the biomedical field. Dendrimers synthesized with mannose at the end group and with entrapped zero-valent copper/silver could be a potential candidate against bacterial proliferation. This study is aimed at investigating the bactericidal activity of metal-glycodendrimers. The Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction was used to synthesize a new mannosylated dendrimer containing 12 mannopyranoside residues in the periphery. The enterotoxigenic Escherichia coli fimbriae 4 (ETEC:F4) viability, measured at 600 nm, showed the half-inhibitory concentration (IC50) of metal-free glycodendrimers (D), copper-loaded glycodendrimers (D:Cu) and silver-loaded glycodendrimers (D:Ag) closed to 4.5 × 101, 3.5 × 101 and to 1.0 × 10−2 µg/mL, respectively, and minimum inhibitory concentration (MIC) of D, D:Cu and D:Ag of 2.0, 1.5 and 1.0 × 10−4 µg/mL, respectively. The release of bacteria contents onto broth and the inhibition of ETEC:F4 biofilm formation increased with the number of metallo-glycodendrimer materials, with a special interest in silver-containing nanomaterial, which had the highest activity, suggesting that glycodendrimer-based materials interfered with bacteria-bacteria or bacteria–polystyrene interactions, with bacteria metabolism and can disrupt bacteria cell walls. Our findings identify metal–mannose-dendrimers as potent bactericidal agents and emphasize the effect of entrapped zero-valent metal against ETEC:F4.


Introduction
Enterotoxigenic Escherichia coli (ETEC) bacteria are the main cause of enteric swine diseases [1] and were intensively studied in porcine post-weaning diarrhea characterized by growth retardation of piglets, dysbiosis, hemorrhagic diarrhea, pathogenic infections due to hyperpermeability of enterocytes or sudden death, causing economic upheaval in pig production [2][3][4].The ETEC bacteria were found in pig foods, slaughterhouses, or contaminated instruments in contact with the animals.The prophylactic strategies to prevent bacterial infections are based on cleaning and sterilizing the swine environment.Fecal contamination is the main vehicle for zoonotic pathogens [5][6][7][8].Several pathogenic bacteria, including ETEC, found in livestock can contaminate water, soil and farm environments and cause disease in humans and pets [9,10].ETEC are characterized by the ability to produce two types of virulent factors: adhesins promoting binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion [11].During the disease, the proliferation of ETEC induces a disequilibrium of the commensal microbiome [12][13][14] and generates severe hemorrhagic diarrhea in mammals that may cause inappetence, loss of weight and even sudden death [15].The development and administration of vaccines [16][17][18][19], bactericidal [20,21] and microbiota transplantation [22] remain the main therapeutic strategies against ETEC infection.In the last decade, many cases of antibacterial resistance were reported [23] that have shown the importance of engineering novel antibacterial drugs.In this project, we proposed glycodendrimer-based materials.Glycodendrimers are starburst polymers with predictable molecular weight and organized with a central core surrounded by ramifications ended with reactive groups [24].Due to the hyperbranched structure and the presence of internal cavities, glycodendrimers are used as carriers for bioactive agents such as drugs or metallic ions.In addition, bioactive molecules (cationic, anionic, polar and non-polar) can be attached at the periphery of the glycodendrimer via the reactive terminal groups.The reactive group could interact with various therapeutic molecules such as DNA, proteins, carbohydrates, or metals according to the biological applications [25].Further, multivalency is a real advantage for glycodendrimers compared to carbohydrate monomers [26].Indeed, multivalent carbohydrate-protein target interactions have shown significant advantages, compared to the interactions between carbohydrate monomers and protein targets, due to low binding affinity [27].For this purpose, several glycodendrimers have been designed to solve the problem of low-affinity carbohydrate-protein interactions [28].Mannosylated analogs [29] glycodendrimers are considered potential candidates for the treatment of certain strains of E. coli by inhibiting bacterial adhesion and the formation of biofilms on the cell surface [30].Further biological applications of glycodendrimers have been reported [31][32][33].
Due to their anti-fungal and antibacterial properties, glycodendrimers are usually used to inhibit bacterial proliferation [34]; therefore, they constitute a non-neglected route to control bacterial proliferation.Other biopolymers, suitable as carriers for silver and copper are already used in the laboratory to control Gram-negative bacterial proliferation.It was previously shown [35,36] that zero-valent silver and copper hosted by carboxymethyl derivatives are bactericides against non-pathogenic Gram-negative bacteria.We propose now an investigation of the bactericidal activity of glycodendrimers unloaded and loaded zero-valent metals.The mechanism of action of glycodendrimers is not yet completely understood.It is known that certain glycodendrimers carrying metal cations have a hydrophobic tail able to penetrate the cell or facilitate their attachment to the bacterial wall [37,38], changing the membrane permeability by making it porous, and leading to cell lysis.The positive charge of metal cations or of the carbohydrate terminal group and the amphiphilicity alongside the dynamic self-assembling of glycodendrimers are enrolled in their antibacterial activity [39][40][41][42][43][44].The appearance of pores on the membrane may allow the diffusion of glycodendrimers into the cell, where the carbohydrate terminal groups bind with sulfur and phosphorus-containing proteins, leading to their inactivation and with DNA [45].Another hypothesis suggests that the antibacterial activity of glycodendrimer loaded with metal nanoparticles results from the release of corresponding ions via the oxidation dissolution process.Metal ions oxidized from corresponding metal-loaded materials mainly interact with thiol groups of various enzymes and proteins, thereby interfering with the respiratory chain and disrupting the bacterial cell wall [46].In addition, it is known that silver ions are involved in the generation of reactive oxygen species (ROS), which are considered the main cause of most cell deaths via the inactivation of DNA replication and ATP production [47].In the same way, it was also found that several dendrimers possess antibacterial activity against pathogenic E. coli [48].
Metallo-glycodendrimer materials combining the nanoparticle properties due to their nanosize and the alteration of bacteria metabolism due to mannose are expected to be good candidates to overcome microbial proliferation.To the best of our knowledge, there are no reports on the synthesis and on the antibacterial applications of mannosylated dendrimers loaded with zero-valent copper or silver.There are now reports of the synthesis of novel glycodendrimers loaded with zero-valent metals and their applications against enterotoxigenic E. coli proliferation (the main cause of swine enteritis).The core of dendrimers was prepared using gallic acid, which has intrinsic antibacterial properties.Mannopyranosides, known to target bacterial pili, were attached as ending reactive groups to form glycodendrimers.In order to enhance the antibacterial activity of these glycodendrimers, zero-valent copper or silver was loaded to form copper-loaded glycodendrimers and silver-loaded glycodendrimers, respectively.The particle size of glycodendrimers-based materials was measured, zero-valent metals entrapped was confirmed, and the antimicrobial activity of metal-free glycodendrimers, copper-loaded glycodendrimers and silver-loaded glycodendrimers against ETEC:F4 was compared with that of 3% hydrogen peroxide.

Materials
All the reagents were used as supplied without any prior purification.The reagents were obtained from Millipore Sigma Canada Ltd. (Oakville, ON, Canada) and Thermo Fisher Scientific (Saint-Laurent, QC, Canada).Enterotoxigenic E. coli Fimbriae 4 (NCBI ID: txid316401), used for bactericidal assays, was from Professor Fairbrother (Pathology and Microbiology Department) at the Veterinary Medicine Faculty of Université de Montréal (St-Hyacinthe, QC, Canada).

Methods
All the organic reactions were carried out using standard methods under an inert atmosphere of nitrogen.The storage of the solvents was carried out using molecular sieves and if necessary, those solvents were bubbled with nitrogen.The monitoring of the reactions was carried out by using thin-layer chromatography (TLC) using silica gel 60 F254 pre-coated plates (E.Merck, Darmstadt, Germany).The TLC was viewed under ultraviolet light at 254 nm or/and chemical stain recipe.The purification was performed by recrystallization or flash (60 Å porosity, 40-63 µm) column chromatography using silica gel (Canadian Life Science, Peterborough, ON, Canada).
The distribution of particle size was measured in water using Dynamic Light Scattering measurements (Malvern, Zetasizer Nano S90, Worcestershire, UK).
1 H-NMR acquisitions were recorded at 300 MHz, and 13 C-NMR spectra were recorded at 75 MHz, respectively, on a Bruker spectrometer (300 MHz) (Milton, ON, Canada).All NMR spectra were measured at 25 • C in the described deuterated solvents.The chemical shifts of proton and carbon are reported in parts per million (ppm), and the coupling constants (J) are reported in Hertz (Hz).The peaks of the residual protic solvent used for chemical shift calibrations were CDCl 3 ( 1 H, δ 7.27 ppm; 13 C, δ 77.2 ppm (central resonance of the triplet)), DMSO-d6 ( 1 H, δ 2.50 ppm; 13 C, δ 39.52 ppm) and D 2 O ( 1 H, 4.79 ppm and 30.9 ppm for the CH 3 of the acetone in the 13 C spectra).
Synthesis of methyl 3,4,5-tris(propargyloxy)benzoate (3).Compound 2 (1.0 g, 5.4 mmol) was dissolved in 10 mL of dry acetone to which was added potassium carbonate K 2 CO 3 (5.2g, 43.4 mmol) followed by the addition of 18-crown-6-ether (57.4 mg, 0.2 mmol) as a co-catalyst.Propargyl bromide (1.6 mL, 43.4 mmol) was next added dropwise, and the reaction was refluxed overnight.The solvent was then evaporated to afford the crude product, which was purified by silica gel column chromatography, which gave compound 3 as a white powder (1.10 g, yield 68%); Rf = 0.4 (15% ethyl acetate/hexane).Compound characterization agreed with literature values [49]; 1  Synthesis 3,4,5-tris(propargyloxy)benzoic acid (4).Compound 3 (1.0 g, 3.4 mmol) was dissolved in 40 mL ethanol, followed by the addition of a 10% aqueous solution of KOH (7.5 mL, 752.4 mg, 13.4 mmol) in 7.5 mL water.The reaction was refluxed for 4 h with constant stirring and cooled to room temperature.Subsequently, the reaction mixture was concentrated, and hydrochloric acid was added until pH 1 was obtained.The reaction mixture was extracted with dichloromethane (DCM) and washed with H 2 O.The organic layer was then collected and dried over Na 2 SO 4, followed by the solvent evaporation without purification to provide pure compound 4 as a white powder (905 mg, yield 95%); Rf = 0.4 (15% ethyl acetate/hexane).Compound characterization agreed with literature values [49]  Synthesis of dendrimer core (5).To a solution of pentaerythritol (25.0 mg, 0.18 mmol) in 10 mL anhydrous DCM (10 mL), compound 4 (271.4mg, 0.95 mmol), N,N ′dicyclohexylcarbodiimide (DCC) (189.4 mg, 0.92 mmol) and 4-dimethylaminopyridine DMAP (49.4 g, 0.40 mmol) were added.The reaction was refluxed overnight (o.n.).The completion of the reaction was confirmed by TLC, and the reaction mixture was concentrated and purified by silica gel column chromatography to afford compound 5 as a white solid (146.8 mg, yield 67%); Rf = 0.4 (DCM); 1  Synthesis of triethylene glycol p-toluenesulfonate (6).To a solution of triethylene glycol (16.24 g, 108.15 mmol) in 45 mL of tetrahydrofuran (THF), 6 mL of a 4M aqueous solution of NaOH was added.The reaction mixture was stirred at 0 • C for 1 h, and then a solution of tosyl chloride (2.11 g, 10.82 mmol) in THF (25 mL) was added dropwise using a dropping funnel.Finally, the reaction mixture was stirred at 0 • C for an additional 3 h.The reaction mixture was poured into iced water (200 mL) and extracted with DCM (3 × 200 mL).The organic layer was dried over sodium sulphate and concentrated under vacuo.The crude product was purified by silica gel column chromatography to give compound 6 as a colorless oil (3.29 g, 99%) Rf = 0.4 (30% acetone/DCM).Compound characterization agreed with the literature value [50]. 1
Effect of glycodendrimers on ETEC proliferation.Enterotoxigenic E. coli fimbriae 4 (ETEC:F4) 1 × 10 7 CFU/mL were treated with various concentrations of metal-glycodendrimers (0-0.075mg/mL of broth) loaded or not with metal nanoparticles.For the negative control, the ETEC:F4 bacteria were treated with Luria-Bertani (LB) broth from Becton, Dickinson and Company (Sparks, MD, USA).The samples at a final volume of 10 mL were incubated for 24 h at 37 • C, 100 RPM and the optical density at 600 nm (OD 600nm ) was measured in a polystyrene cuvette of 10 mm path length by Biochrom Libra S50 UV-Vis spectrophotometer (Biochrom US, Holliston, MA, USA).The bactericidal activity of metal-glycodendrimers was also evaluated by inhibition zone diameter of 1 mg powder of glycodendrimers on LB agar Petri dishes, previously inoculated with ETEC:F4.The disc images were acquired after 24 h incubation at 37 • C, analyzed by ImageJ2 software (version 2, Madison, WI, USA) and the diameters of the inhibition zones were reported in centimeters.
Quantification of protein released by ETEC:F4 treated with metal-loaded glycodendrimers for 24 h at 100 RPM.In order to understand the metallo-glycodendrimers mechanisms on ETEC:F4 death, proteins, DNA, RNA and oligonucleotides released from bacteria were measured in the LB broth.A volume of 5 mL was taken from the mixture of bacteria previously incubated with corresponding glycodendrimers for 24 h.The sample was centrifugated at 800 RPM × 10 min at 4 • C, and the supernatant was collected to quantify bacteria-released contents.The Bradford method [53] was used to assay proteins contained in the supernatant by absorbance measurement at 595 nm.
DNA, RNA and oligonucleotide assay.A volume of 0.5 mL of the supernatant collected from bacteria culture centrifugation (800 RPM × 10 min at 4 • C) was transferred to spin columns (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada) inserted into a 2 mL Eppendorf used as a collection tube and centrifuged at 6000×g for 2 min at 4 • C according to the protocols of nucleic acids separation of the manufacturer.The centrifugation rounds were repeated three times.For each round, new spin columns and new Eppendorf vials are used to collect the elution solutions.The DNA, RNA and oligonucleotide concentrations were acquired by NanoDrop™ 2000/2000c Spectrophotometer (Thermo Fisher Scientific, Mississauga, ON, Canada) based on the 260/280 or 260/230 ratios following the protocols of the manufacturer.
Biofilm assay.ETEC was identified as a biofilm-forming species.Biofilm is a critical factor for microbial survival and antibiotic resistance.ETEC:F4 biofilm formation was followed in polystyrene 96-well plates.Bacteria, at a concentration of 10 7 CFU/mL treated or not with glycodendrimer-based materials 0.005 mg/mL (a concentration close to the IC 50 of less toxic glycodendrimeric materials), were grown at 37 • C for 24 h in Luria-Bertani broth.After incubation, bacteria were gently washed three times with 200 µL of PBS in order to remove non-adhered bacteria.The attached bacteria, representing the matrix components of biofilm, were stained with 100 µL of a 0.1% solution of crystal violet for 30 min at room temperature, according to Mintzer et al.'s protocol [54].Crystal violet salt was then solubilized by the addition of 30% glacial acetic acid and incubated at room temperature for 10-15 min.The absorbance was measured by a Biochrom EZ Read 800 microplate reader (Biochrom US, Holliston, MA, USA) at 570 nm, and data were reported as mean ± standard deviation.All experiments were repeated three times.

Results and Discussion
The glycodendrimer was conceived as based on antibacterial constituent elements exhibiting inner antibacterial activity.Thus, gallic acid, which is a natural polyphenol that is found in plants, was chosen for the synthesis route of the glycodendrimer core.Gallic acid is known to have strong antibacterial properties on its own and can affect irreversibly the E. coli membrane [55].Additionally, mannose was also chosen because it has been well established that E. coli possesses a carbohydrate-binding protein at the tip of their pili associated with FimH, which recognizes α-D-mannopyranoside glycoconjugates on the host cell membranes [29].Hence, mannoside NPs can be envisaged as targeting devices.Therefore, combining the above two components into a single entity presented into a multivalent architecture (mannosylated glycodendrimer) was hypothesized to greatly enhance fighting E. coli bacterial infections.

Synthesis of the Core Structure
The propargylated core structure was built from pentaerythritol and a propargylated gallic acid derivative (Scheme 1).First, methyl gallate (2) [49] was obtained from the 3,4,5trihydroxybenzoic by using Fischer esterification in order to protect the acid functionality.Then, the methyl ester was functionalized with alkyne groups in the periphery, and the propargylation reaction, including phenol groups, led to compound 3.Then, the obtained compound 3 was treated with aqueous KOH to hydrolyze the ester and give the compound 4 (3,4,5-tris(propargyloxy)benzoic acid) (Scheme 1) [56].
Microorganisms 2024, 12, 966 7 of 17 the E. coli membrane [55].Additionally, mannose was also chosen because it has been well established that E. coli possesses a carbohydrate-binding protein at the tip of their pili associated with FimH, which recognizes -D-mannopyranoside glycoconjugates on the host cell membranes [29].Hence, mannoside NPs can be envisaged as targeting devices.Therefore, combining the above two components into a single entity presented into a multivalent architecture (mannosylated glycodendrimer) was hypothesized to greatly enhance fighting E. coli bacterial infections.

Synthesis of the Core Structure
The propargylated core structure was built from pentaerythritol and a propargylated gallic acid derivative (Scheme 1).First, methyl gallate (2) [49] was obtained from the 3,4,5trihydroxybenzoic by using Fischer esterification in order to protect the acid functionality.Then, the methyl ester was functionalized with alkyne groups in the periphery, and the propargylation reaction, including phenol groups, led to compound 3.Then, the obtained compound 3 was treated with aqueous KOH to hydrolyze the ester and give the compound 4 (3,4,5-tris(propargyloxy)benzoic acid) (Scheme 1) [56].
Scheme 1. Synthesis of the key dendritic pentaerythritol scaffold harboring four gallic acid residues covered with 12 propargyl functions used for the preparation of multivalent mannosylated dendrimer by a copper-catalyzed azide-alkyne cycloaddition (CuAAC).
A dendrimer scaffold harboring 12 propargyl groups was chosen in order to allow a first generation with 12 mannoses on the glycodendrimer periphery, so 48 hydroxyl groups only for generation 1, which is remarkable given that the comparable number of hydroxyl groups is generally obtained from higher generations for commercial poly(amidoamine) dendrimers [57].The propargylated gallic acid (4) was reacted according to the Scheme 1. Synthesis of the key dendritic pentaerythritol scaffold harboring four gallic acid residues covered with 12 propargyl functions used for the preparation of multivalent mannosylated dendrimer by a copper-catalyzed azide-alkyne cycloaddition (CuAAC).
A dendrimer scaffold harboring 12 propargyl groups was chosen in order to allow a first generation with 12 mannoses on the glycodendrimer periphery, so 48 hydroxyl groups only for generation 1, which is remarkable given that the comparable number of hydroxyl groups is generally obtained from higher generations for commercial poly(amidoamine) dendrimers [57].The propargylated gallic acid (4) was reacted according to the Steglich reaction with pentaerythritol using DCC as a coupling agent and Dimethyl aminopyridine (DMAP) as a nucleophile to afford the tetravalent glycodendrimer core (5) by esterification coupling.According to the literature, the Steglich reaction [58] carried out at room temperature is known to take several days.The 1 H-NMR confirmed the presence of the propargylic protons at around 2.5 ppm (Figure S1), and the product was confirmed by 13 C-NMR (Figure S2).

Synthesis of the Carbohydrate for Core Branching
For the sugar moiety, our approach aimed to synthesize an ethylene glycol linker using triethylene glycol: a Food and Drug Administration (FDA)-approved agent.A monotosylation of triethylene glycol was carried out under cold conditions to obtain the desired product without any by-product.The D-mannose was also treated with acetic anhydride in pyridine for the acetylation of the mannose hydroxyl groups to afford the 1,2,3,4,6penta-O-acetyl-α/β-D-mannopyranose (7) which was glycosylated with triethylethylene glycol p-toluenesulfonate (6) using Lewis acid (BF 3 -Et 2 O) to give the 2-(2-(2-(2-Tosyloxy-ethoxy)-ethoxy)-ethyl)2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (8).Finally, by carrying out nucleophilic substitution, the tosylate group of compound 8 was converted into an azide group present at the focal point of our linker.(Scheme 2). 1 H-NMR showed the disappearance of the aromatic protons of the tosylate group at 7.77 and 7.32 ppm, as expected.
For the sugar moiety, our approach aimed to synthesize an ethylene glycol linker using triethylene glycol: a Food and Drug Administration (FDA)-approved agent.A monotosylation of triethylene glycol was carried out under cold conditions to obtain the desired product without any by-product.The D-mannose was also treated with acetic anhydride in pyridine for the acetylation of the mannose hydroxyl groups to afford the 1,2,3,4,6-penta-O-acetyl-α/β-D-mannopyranose (7) which was glycosylated with triethylethylene glycol p-toluenesulfonate (6) using Lewis acid (BF3-Et2O) to give the 2-(2-(2-(2-Tosyloxy-ethoxy)-ethoxy)-ethyl)2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (8).Finally, by carrying out a nucleophilic substitution, the tosylate group of compound 8 was converted into an azide group present at the focal point of our linker.(Scheme 2). 1 H-NMR showed the disappearance of the aromatic protons of the tosylate group at 7.77 and 7.32 ppm, as expected.

Synthesis of Mannosylated Glycodendrimer
The Copper-assisted Azide-Alkyne Cycloaddition (CuAAC), according to Sharpless et al. [55,59] is known to be efficient, simple, selective and frequently used in dendrimer synthesis.
The 1 H-NMR of 10 gives some indications of the reaction accomplishment.First, the disappearance of the two asymmetrical propargyl proton peaks at δ 2.61 ppm and δ 2.47 ppm, as well as the appearance of the triazole peaks at δ 8.01 and δ 7.99 ppm, unambiguously confirmed the completion of the click reaction (Figure S3).The 1 H-NMR analysis was also supported by 13 C-NMR (Figure S4).The deprotection of the peracetylated glycodendrimer 10 (Scheme 3) was carried out under mild Zemplén trans-esterification conditions (NaOMe, MeOH, pH 8.5), which allowed maintaining the inner (protected) gallate esters.The whole disappearance of the acetate peaks (144 protons) in the 1 H-NMR spectra of the resulting mannodendrimer 11 (Scheme 3) appeared between δ 1.9 and 2.2 ppm (Figure S5).The unprotected glycodendrimer (11) identified by 13 C-NMR (Figure S6) spectra confirmed the completion of the reaction.

Synthesis of Mannosylated Glycodendrimer
The Copper-assisted Azide-Alkyne Cycloaddition (CuAAC), according to Sharpless et al. [55,59] is known to be efficient, simple, selective and frequently used in dendrimer synthesis.
The 1 H-NMR of 10 gives some indications of the reaction accomplishment.First, the disappearance of the two asymmetrical propargyl proton peaks at δ 2.61 ppm and δ 2.47 ppm, as well as the appearance of the triazole peaks at δ 8.01 and δ 7.99 ppm, unambiguously confirmed the completion of the click reaction (Figure S3).The 1 H-NMR analysis was also supported by 13 C-NMR (Figure S4).The deprotection of the peracetylated glycodendrimer 10 (Scheme 3) was carried out under mild Zemplén trans-esterification conditions (NaOMe, MeOH, pH 8.5), which allowed maintaining the inner (protected) gallate esters.The whole disappearance of the acetate peaks (144 protons) in the 1 H-NMR spectra of the resulting mannodendrimer 11 (Scheme 3) appeared between δ 1.9 and 2.2 ppm (Figure S5).The unprotected glycodendrimer (11) identified by 13 C-NMR (Figure S6) spectra confirmed the completion of the reaction.
Microorganisms 2024, 12, 966 9 of 17 Scheme 3. Synthesis of mannosylated glycodendrimer 11 which is endowed with 12 α-D-mannopyranoside residues at the periphery.The synthesis was accomplished according to the well-established "click chemistry" (CuAAC) between the propargylated core 5 and the mannosylated sugar azide 9, followed by a selective Zemplén reaction.

Characterization of Nanoparticles of Cu-and Ag-Loaded Glycodendrimers
The relevant antibacterial properties of zero-valent copper and silver nanoparticles hosted by biopolymers are already documented [35] and these zero-valent metals were used in this project to increase the antibacterial activity of the glycodendrimers.The metal ions from Cu(OAc)2 and AgNO3 were first uniformly distributed in the dendrimeric dispersion and then reduced with NaBH4 to generate the corresponding zero-valent metals, homogenously entrapped by glycodendrimers.A DLS study of metal-glycodendrimeric Scheme 3. Synthesis of mannosylated glycodendrimer 11 which is endowed with 12 α-Dmannopyranoside residues at the periphery.The synthesis was accomplished according to the well-established "click chemistry" (CuAAC) between the propargylated core 5 and the mannosylated sugar azide 9, followed by a selective Zemplén reaction.

Characterization of Nanoparticles of Cu-and Ag-Loaded Glycodendrimers
The relevant antibacterial properties of zero-valent copper and silver nanoparticles hosted by biopolymers are already documented [35] and these zero-valent metals were used in this project to increase the antibacterial activity of the glycodendrimers.The metal ions from Cu(OAc) 2 and AgNO 3 were first uniformly distributed in the dendrimeric dispersion and then reduced with NaBH 4 to generate the corresponding zero-valent metals, homogenously entrapped by glycodendrimers.A DLS study of metal-glycodendrimeric materials showed particle sizes smaller than 100 nm for the metal-loaded glycodendrimers.The medium size for the D:Cu was slightly lower (41-64 nm) than for the D:Ag (61-80 nm), whereas the medium size of unloaded glycodendrimers (D) was 64-65 nm, suggesting no major impact of metal loading on the size of dendrimers.In addition, polydispersity indexes of 0.42 ± 0.03, 0.47 ± 0.03 and 0.33 0.01 were obtained for D, D:Cu and D:Ag, respectively.

Bactericidal Activity of Metal-Glycodendrimers
The enterotoxigenic Escherichia coli fimbriae 4 (ETEC:F4) was selected to evaluate the bactericidal effect of zero-valent metal entrapment in glycodendrimers.The decrease in ETEC survival measured by optical density at 600 nm was inversely proportional to the increase in glycodendrimer concentrations (Figure 1).The minimal inhibitory concentrations (MIC) were 2.0, 1.5 and 1.0 × 10 −4 µg/mL for glycodendrimers (D), glycodendrimers loaded with copper (D:Cu) and glycodendrimers loaded with silver (D:Ag), respectively.These values were markedly lower (10-1000 folds) than MIC of anti-ETEC:F4 agents used in veterinary and human medicine.In the same way, the half inhibitory concentrations of bacteria growth (IC 50 ) were 4.5 × 10 1 , 3.5 × 10 1 and 1.0 × 10 −2 µg/mL for D, D:Cu and D:Ag, respectively (Figure 2, inserted table).These data support our hypothesis that the addition of mannose (antimicrobial carbohydrate) as terminal groups and the loading of glycodendrimers by zero-valent copper and silver nanoparticles, inhibited the ETEC:F4 proliferation and enhanced the bactericidal activity of designed dendrimer, with the highest activity for silver zero loaded glycodendrimers (Table S1).
proliferation and enhanced the bactericidal activity of designed dendrimer, with the highest activity for silver zero loaded glycodendrimers (Table S1).The inhibition of ETEC:F4 bacteria proliferation at metal glycodendrimer-dependent concentrations (Figure 1) correlated with the inhibition diameters (Figure 2A,B) confirmed the bactericidal activity of glycodendrimeric materials loaded or not with zero-valent metal (Table S1).Additional experiments were conducted with Phosphomycin, Gentamycin and Kanamycin and compared with our unloaded (D) and metal-loaded mannodendrimers (D:Cu°, D:Ag°).The choice of Phosphomycin, Gentamycin and Kanamycin was based on the fact that these antibiotics are currently used to treat enterotoxigenic E. coli The inhibition of ETEC:F4 bacteria proliferation at metal glycodendrimer-dependent concentrations (Figure 1) correlated with the inhibition diameters (Figure 2A,B) confirmed the bactericidal activity of glycodendrimeric materials loaded or not with zero-valent metal (Table S1).Additional experiments were conducted with Phosphomycin, Gentamycin and Kanamycin and compared with our unloaded (D) and metal-loaded mannodendrimers (D:Cu • , D:Ag • ).The choice of Phosphomycin, Gentamycin and Kanamycin was based on the fact that these antibiotics are currently used to treat enterotoxigenic E. coli (ETEC).The results expressed as diffusion diameters showed our materials D:Ag with a moderately higher bactericidal efficacy than those of Phosphomycin and Gentamycin and comparable with that of Kanamycin.Differently, the D and D:Cu materials presented a lower bactericidal efficacy (Table S1).
At concentrations of 10 mM and higher, H 2 O 2 may react with DNA and other macromolecules of bacteria and generate the highly reactive and damaging hydroxyl radical (HO•) via the Fenton reaction [60].The choice of 3% H 2 O 2 (~1M) as positive control is based on the fact that, at this much higher concentration (around 100 times greater than that mentioned as cytotoxic), it is currently used as a disinfectant in slaughterhouses and for farm equipment due to its potential to kill all kinds of cells [61,62] including E. coli [60,63,64].
A common mechanism of antibacterial agents is the disruption of bacterial walls or the modification of nucleotidic contents.In order to confirm the metal glycodendrimer action on the loss of integrity of bacteria wall, the proteins released in the LB broth from ETEC:F4 treated or not with various concentrations of metal-glycodendrimer (0-0.03mg/mL) for 24 h was assayed by Bradford method [53] based on the absorbance measurement at 595 nm, whereas DNA, RNA and oligonucleotide concentrations were acquired according to the protocol of the nanodrop spectrophotometer manufacturer.
To quantify released proteins (Figure 3A), DNA (Figure 3B), RNA (Figure 3C) and oligonucleotides (Figure 3D), the ETEC:F4 (1 × 10 7 CFU/mL) was treated with various concentrations (0-0.03mg/mL) of glycodendrimer-based materials.It was found that the bacterial cytoplasmic content increases in LB broth with the concentration of glycodendrimeric materials (Figure 3), confirming a loss of integrity of ETEC:F4 membrane during the treatment and the bactericidal activity of glycodendrimers (D), copper-loaded glycodendrimers (D:Cu) and silver-loaded glycodendrimers (D:Ag).The capacity to form biofilm was investigated and compared between the different glycodendrimeric materials using the crystal violet method indirectly related to the presence of colored solution with absorbency at 570 nm.Treatment of bacteria with 0.005 mg/mL D, D:Cu and D:Ag inhibited the capacity to form the biofilm in comparison to the untreated ETEC:F4 (Figures 4 and S7).The absorbency resulting from the reaction of crystal violet and the biofilm generated by bacteria grown with D:Cu and D:Ag was substantially reduced by approximately 20, 30 and 70%, respectively, when compared the untreated (blank), considered as 100%.Supposing a loss of ETEC membrane integrity, the treatment with D, D:Cu and D:Ag generated an increase in protein concentration in LB broth, but this was not proportional to the increasing concentrations of glycodendrimeric materials.The plot obtained from the releasing of proteins versus the concentration of the glycodendrimers (0-0.03mg/mL) represented two phases: the first one faster, with a high slope, correlated with a higher amount of ETEC:F4 in the LB broth and with an unstable rate of protein release from bacteria, and the second slower phase, with a low or moderately low slope, which might be related to protein release at a stable rate.
Coomassie brilliant blue R250 contains two negatively charged sulphated groups, able to establish electrostatic interactions with cationic amino acids of proteins.No metaldependent interference was found between Coomassie R250 dye with Cu(OAc) 2 and AgNO 3 salts, or with copper-and silver-loaded glycodendrimers.So, proteins were accurately detected in the broth and were from bacteria.
The values of bioactive agents obtained with the 3% H 2 O 2 positive control were constant for each sample due to the identical concentration.Supposing that each of the three glycodendrimers might disrupt the ETEC wall and liberate its content into the LB broth, the bactericidal effects of each glycodendrimer were also evaluated by assays of released DNA, RNA and oligonucleotides.Among the investigated agents, D:Ag appeared, as expected, to be the most bactericidal material.In opposite to our attempts, the DNA and oligonucleotides released from bacteria cytosol by D:Ag treatment was the lowest, suggesting interactions between D:Ag and DNA and between D:Ag and oligonucleotides as observed with many antibacterial agents able to interact and modify nucleic acids and oligonucleotides of bacteria [65].
The capacity to form biofilm was investigated and compared between the different glycodendrimeric materials using the crystal violet method indirectly related to the presence of colored solution with absorbency at 570 nm.Treatment of bacteria with 0.005 mg/mL D, D:Cu and D:Ag inhibited the capacity to form the in comparison to the untreated ETEC:F4 (Figure 4 and Figure S7).The absorbency resulting from the reaction of crystal violet and the biofilm generated by bacteria grown with D, D:Cu and D:Ag was substantially reduced by approximately 20, 30 and 70%, respectively, when compared to the untreated (blank), considered as 100%.These results corroborated those obtained for bactericidal activity assay (Figure 1), suggesting that D, D:Cu and D:Ag might inhibit the bacteria-to-bacteria interaction, interfere with bacteria information transmission, promote bacteria aggregation or prevent adhesion of bacteria to the surface of the well plate [66][67][68].

Conclusions
This report showed the bactericidal activity of metal glycodendrimers formed with gallic acid in the core and containing mannose as the carbohydrate-reactive terminal group.This glycodendrimeric material can host zero-valent metals (Cu 0 and Ag 0 ) and appears as a potent antibacterial candidate.More precisely, this type of glycodendrimer showed activity against enterotoxigenic E. coli fimbriae 4 (ETEC:F4) bacteria with MIC lower than those of several common antibiotics.The entrapment of zero-valent metal increased the bactericidal effect of glycodendrimeric materials, and the Ag-loaded glycodendrimer (D:Ag) was the most potent bactericidal agent compared to Cu-loaded glycodendrimer (D:Cu) and metal-free glycodendrimer (D).These results show the synthesized glycodendrimeric materials as new bactericidal agents against Gram-negative bacteria such as enterotoxigenic E. coli Fimbriae 4. The bactericidal activity could be explained by bacteria wall disruption and by the release of bacteria content in the culture broth.In addition, D, D:Cu and D:Ag were able to inhibit the ETEC:F4 capacity to form biofilm.The bactericidal effects of D:Ag were higher than those of phosphomycin and gentamycin, currently used antibiotics.These results corroborated those obtained for bactericidal activity assay (Figure 1), suggesting that D, D:Cu and D:Ag might inhibit the bacteria-to-bacteria interaction, interfere with bacteria information transmission, promote bacteria aggregation or prevent adhesion of bacteria to the surface of the well plate [66][67][68].

Conclusions
This report showed the bactericidal activity of metal glycodendrimers formed with gallic acid in the core and containing mannose as the carbohydrate-reactive terminal group.This glycodendrimeric material can host zero-valent metals (Cu 0 and Ag 0 ) and appears as a potent antibacterial candidate.More precisely, this type of glycodendrimer showed activity against enterotoxigenic E. coli fimbriae 4 (ETEC:F4) bacteria with MIC lower than those of several common antibiotics.The entrapment of zero-valent metal increased the bactericidal effect of glycodendrimeric materials, and the Ag-loaded glycodendrimer (D:Ag) was the most potent bactericidal agent compared to Cu-loaded glycodendrimer (D:Cu) and metalfree glycodendrimer (D).These results show the synthesized glycodendrimeric materials as new bactericidal agents against Gram-negative bacteria such as enterotoxigenic E. coli Fimbriae 4. The bactericidal activity could be explained by bacteria wall disruption and by the release of bacteria content in the culture broth.In addition, D, D:Cu and D:Ag were able to inhibit the ETEC:F4 capacity to form biofilm.The bactericidal effects of D:Ag were higher than those of phosphomycin and gentamycin, currently used antibiotics.

Figure 1 .Figure 1 .
Figure 1.Effect of metal-glycodendrimers on ETEC:F4 bacteria.Evaluation by OD at 600 nm of survival of enterotoxigenic E. coli fimbriae 4 (ETEC:F4) from LB broth containing glycodendrimerbased compounds at different concentrations.Data are triplicate of three different experiments and are represented by mean ± SD.

Figure 1 .
Figure 1.Effect of metal-glycodendrimers on ETEC:F4 bacteria.Evaluation by OD at 600 nm of survival of enterotoxigenic E. coli fimbriae 4 (ETEC:F4) from LB broth containing glycodendrimerbased compounds at different concentrations.Data are triplicate of three different experiments and are represented by mean ± SD.

Figure 2 .
Figure 2. (A) Diffusimetric profile of the Anti-ETEC:F4 materials.Effect of glycodendrimer (D), of glycodendrimer complexed with copper (D:Cu) and of glycodendrimer complexed with silver (D:Ag) was evaluated on agar-LB gel.A solution of 3% H2O2 was used as a positive control.(B) Average diffusion diameters as growth inhibition of bacteria induced by 1 mg of glycodendrimerbased materials and by 10 L of 3% H2O2.The experiments were repeated three times, and data represented mean ± SD.

Figure 2 .
Figure 2. (A) Diffusimetric profile of the Anti-ETEC:F4 materials.Effect of glycodendrimer (D), of glycodendrimer complexed with copper (D:Cu) and of glycodendrimer complexed with silver (D:Ag) was evaluated on agar-LB gel.A solution of 3% H 2 O 2 was used as a positive control.(B) Average diffusion diameters as growth inhibition of bacteria induced by 1 mg of glycodendrimer-based materials and by 10 µL of 3% H 2 O 2 .The experiments were repeated three times, and data represented mean ± SD.

Figure 3 .
Figure 3. Quantification of released bioactive ETEC:F4 contents.Bacteria were treated with D, D:Cu, D:Ag or with 3% H2O2 (added in the absence of glycodendrimeric materials).The quantification of protein (A) was carried out by the Bradford method, whereas DNA (B), RNA (C) and oligonucleotides (D) concentrations were acquired by the protocol of the manufacturer of NanoDrop spectrophotometer (n = 3, mean ± SD).

Figure 3 .
Figure 3. Quantification of released bioactive ETEC:F4 contents.Bacteria were treated with D, D:Cu, D:Ag or with 3% H 2 O 2 (added in the absence of glycodendrimeric materials).The quantification of protein (A) was carried out by the Bradford method, whereas DNA (B), RNA (C) and oligonucleotides (D) concentrations were acquired by the protocol of the manufacturer of NanoDrop spectrophotometer (n = 3, mean ± SD).