Engineering of a New Bisphosphonate Monomer and Nanoparticles of Narrow Size Distribution for Antibacterial Applications.

In recent years, many bacteria have developed resistance to commonly used antibiotics. It is well-known that calcium is essential for bacterial function and cell wall stability. Bisphosphonates (BPs) have high affinity to calcium ions and are effective calcium chelators. Therefore, BPs could potentially be used as antibacterial agents. This article provides a detailed description regarding the synthesis of a unique BP vinylic monomer MA-Glu-BP (methacrylate glutamate bisphosphonate) and polyMA-Glu-BP nanoparticles (NPs) for antibacterial applications. polyMA-Glu-BP NPs were synthesized by dispersion copolymerization of the MA-Glu-BP monomer with the primary amino monomer N-(3-aminopropyl)methacrylamide hydrochloride (APMA) and the cross-linker monomer tetra ethylene glycol diacrylate, to form cross-linked NPs with a narrow size distribution. The size and size distribution of polyMA-Glu-BP NPs were controlled by changing various polymerization parameters. Near-infrared fluorescent polyMA-Glu-BP NPs were prepared by covalent binding of the dye cyanine7 N-hydroxysuccinimide to the primary amino groups belonging to the APMA monomeric units on the polyMA-Glu-BP NPs. The affinity of the near-infrared fluorescent polyMA-Glu-BP NPs toward calcium was demonstrated in vitro by a coral model. Cytotoxicity, cell uptake, and antibacterial properties of the polyMA-Glu-BP NPs against two common bacterial pathogens representing Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, and two representing Gram-positive bacteria, Listeria innocua and Staphylococcus aureus, were then demonstrated.

Introduction

Bapan class="Chemical">cteria play a major role in pan class="Chemical">causing acute and chronic infections.[1,2] Since the discovery, in 1929, of penicillin by Alexander Fleming,[3] antibiotics have been the preferred treatment for bacterial infections. Unfortunately, in recent years, many bacteria have developed resistance to commonly used antibiotics.[1] Therefore, new effective antibiotics are needed.[2] One possible antibacterial strategy is to reduce the calcium levels, which are essential for the function of bacteria.

Calcium ion forms divalent cation bridges with negatively charged functional groups on the cell wall, which increases bacterial cell stability.[4,5] Calcium also plays an important role in the function of various intracellular proteins in bacteria.[6,7] For example, calcium takes part in the signal transduction of bacteria.[8]

Polyphosphates have been shown to possess antibacterial activity[9−12] due to their ability to form metal complexes with cations such as calcium.[13,14] Polyphosphates are a polymeric multiphosphorous class of compounds bearing a P–O–P repeating unit. Endogenous pyrophosphate is the smallest polyphosphate (Figure ).[15]

Figure 1

Pyrophosphate and BP structures.

pan class="Chemical">Pyrophosphate and pan class="Chemical">BP structures.

pan class="Chemical">Bisphosphonates (pan class="Chemical">BPs) are a group of stable organic analogues of the pyrophosphate (Figure ). BPs have high affinity to calcium and are known as effective calcium chelators.[16−24] Considering the similarity of BPs to polyphosphates, they could be used as antibacterial agents.[25]

Nanoppan class="Chemical">artipan class="Chemical">cles (NPs) are spherical macromolecules with a diameter of 1–100 nm[26] and are being widely studied for their potential as antibacterial drugs.[27,28] Because of their small size, NPs have a relatively large surface-area-to-mass ratio, thus enabling the NPs to interact with the bacterial cell wall without penetrating the cell.[29,30] It has been previously demonstrated that NPs can be active against both Gram-positive and Gram-negative bacteria.[31]

In this work, the synthesis of the novel BP compounds N-phthaloyl glutamate bisphosphonate (N-Pht-Glu-BP), gamma glutamate bisphosphonate (γ-Glu-BP), and MA-Glu-BP (methacrylate glutamate bisphosphonate) is reported. PolyMA-Glu-BP NPs were then synthesized by copolymerization of the novel MA-Glu-BP monomer with two commercially available monomers: (1) N-(3-aminopropyl)methacrylamide hydrochloride (APMA), a monomer containing a primary amine which enables the covalent conjugation of a suitable dye to the NPs, and (2) the cross-linker monomer tetra ethylene glycol diacrylate (TTEGDA). The synthesis and antibacterial properties were investigated. We hypothesize that NPs bearing multiple surface BP units will demonstrate an antibacterial activity.

Results and Discussion

Synthesis of BP Compounds

The synthesis of novel BP compounds N-Pht-Glu-BP, γ-Glu-BP, and MA-Glu-BP is illustrated in Figure . The main challenge of the synthesis of the monomer MA-Glu-BP was the substitution of the BP moiety only on one carboxylic group of the glutamic acid. By synthesizing N-phthaloyl glutamic anhydride, which converts glutamic acid to a cyclic anhydride,[32] the activation of the carboxylic acid in the γ position can be achieved while protecting the carboxylic acid in the α position. The reaction of N-phthaloyl glutamic anhydride with tris(trimethylsilyl)phosphite produced the intermediate N-Pht-Glu-BP,[33] as initially intended. The removal of the N-phthalic protecting group by acidic hydrolysis yielded γ-Glu-BP.[34] The MA-Glu-BP monomer was then obtained by reacting γ-Glu-BP with methacryloyl chloride under basic conditions.[35,36]

Figure 2

Synthesis of the MA-Glu-BP vinylic monomer.

Synthesis of the pan class="Chemical">MA-Glu-BP vinylipan class="Chemical">c monomer.

Synthesis of PolyMA-Glu-BP NPs

Cross-linked polyMA-Glu-BP NPs were synthesized by heterogeneous dispersion co-polymerization of the novel BP monomer MA-Glu-BP with the monomer APMA (which contains a primary amine for the covalent conjugation of a dye to the surface of the NPs) and the cross-linker monomer TTEGDA (Figure A). The obtained spherical polyMA-Glu-BP NPs have a dry diameter of 61.2 ± 6 nm, as measured from the high-resolution transmission electron microscopy (TEM) images (Figure B), and a hydrodynamic diameter of 163.2 ± 7 nm (Figure C). The reason for the dry diameter of the NPs being significantly smaller than the hydrodynamic diameter is probably because the hydrodynamic diameter takes into account the solvent molecules adsorbed on the surface and within the NPs as well as the Brownian motion.[37]

Figure 3

Preparation scheme (A), TEM image (B), and hydrodynamic size histogram (C) of polyMA-Glu-BP NPs.

pan class="Chemical">Preppan class="Chemical">aration scheme (A), TEM image (B), and hydrodynamic size histogram (C) of polyMA-Glu-BP NPs.

Information regarding the stability of the polyMA-Glu-BP NPs was obtained by zeta (ζ)-potential measurements. These measurements were performed by gradually changing the pH from 2.5 to 10.9. Figure exhibits the ζ-potential curve of the dispersed polyMA-Glu-BP NPs (0.1 mg/mL) at different pH values. At pH 2.5, the graph exhibits a low positive ζ-potential, which may be explained by the presence of protonated amine groups on the surface of the NPs. From pH 2.5 to 10.9, the curve shows a significant decrease in the ζ-potential. This behavior is explained by the deprotonation of the BP functional group as well as the deprotonation of the carboxylic acid. The NPs are stable at around pH 7.4 (physiological pH) and exhibit a ζ-potential of −24.3 ± 0.9 mV. The isoelectric point calculated was 2.9.

Figure 4

ζ potential of polyMA-Glu-BP NPs at various pH values.

ζ potential of pan class="Chemical">polyMA-Glu-BP NPs at vpan class="Chemical">arious pH values.

Kinetics of the Formation of the PolyMA-Glu-BP NPs

Kinetics of the formation of the polyMA-Glu-BP NPs was measured by following the change in the hydrodynamic diameter and yield of the formed NPs prepared according to the Experimental Section. Figure A demonstrates a marked decrease in the average diameter of the NPs early on in the polymerization reaction. One minute after initiation of the polymerization, the diameter measured was 557 ± 26 nm, decreasing to 371 ± 28 nm after 2 min and reaching 259 ± 29 nm after 3 min. Then, the diameter of the particles continues to reduce gradually as the reaction progresses, giving diameters of 253 ± 13, 236 ± 17, and 215 ± 19 nm after 5, 15, and 30 min of polymerization, respectively. The NP diameter stabilized after 60 min of polymerization at 183 ± 8 nm. Following 120, 180, 240, 360, 480, 600, and 720 min, the particles present similar diameters: 171 ± 10, 167 ± 6, 167 ± 5, 171 ± 8, 167 ± 7, 163 ± 8, and 160 ± 7 nm, respectively. The NP diameter plateaus at an average of 166 ± 7 nm. This could be explained by the rapid formation of a cross-linked polymer network, which forms the initial particles. As the polymerization progresses, the cross-linkage increases and the NPs become more compact and therefore decrease in diameter.[38−40]

Figure 5

Kinetics of the formation of the polyMA-Glu-BP NPs as measured by following the change in the hydrodynamic diameter (A) and yield (B) of the formed NPs prepared according to the Experimental Section.

Kinetipan class="Chemical">cs of the formation of the pan class="Chemical">polyMA-Glu-BP NPs as measured by following the change in the hydrodynamic diameter (A) and yield (B) of the formed NPs prepared according to the Experimental Section.

To determine the optimal reaction time for the polymerization of the polyMA-Glu-BP NPs, the samples were purified, lyophilized, and weighed. The yield at various time points was calculated (Figure B). It was noted that the majority of the polymerization occurred during the initial 30 min (7.4 ± 3% after 5 min and 28.7 ± 2% after 30 min of polymerization). The yield continued to increase gradually to 29.3 ± 2% after 240 min and to 30 ± 1% after 480 min. A plateau of 32 ± 2% was reached after 720 min of reaction.

Effect of the Polymerization Parameters on the Size and Size Distribution of the Formed PolyMA-Glu-BP NPs

Effect of the Total Monomer Concentration

Figure illustrates the effepan class="Chemical">ct of the increase of the total monomer concentrations on the hydrodynamic diameter and size destitution of the formed polyMA-Glu-BP NPs. The monomers MA-Glu-BP, APMA, and TTEGDA were kept at a constant ratio of 35, 10, and 55 wt %, respectively, but with different total concentrations, while the other reaction conditions remain untouched.

Figure 6

Relationship between the total monomer concentration and the diameter size and size distribution of the formed particles.

Relationship between the total monomer pan class="Chemical">conpan class="Chemical">centration and the diameter size and size distribution of the formed particles.

It is evident, as shown in Figure , that as the total monomer concentration is raised from 1.5 to 5, 7.5, and 10%, there is a consistent increase in the hydrodynamic diameter and size distribution of the formed polyMA-Glu-BP NPs, 133 ± 10 to 242 ± 16, 438 ± 26, and 519 ± 38 nm, respectively. These findings can be attributed to the increasing total amount of polymer formed in the reaction as well as the accelerating polymerization rate. A higher concentration of monomers increases the solubility of the oligomers formed, enabling the formation of longer polymer chains prior to precipitation. In addition, the increase in the concentration leads to agglomeration of the formed particles and therefore an increase in the particle diameter. A similar behavior on the effect of the total monomer concentration on the size and size distribution of various NPs was already reported in the literature.[41−43]

Effect of the Cross-Linker Concentration

Figure displays the influence of increasing the cross-linker TTEGDA concentration on the hydrodynamic diameter and size distribution of the particles (while the weight ratio between MA-Glu-BP and APMA remained untouched).

Figure 7

Effect of the weight % ratio [TTEGDA]/([MA-Glu-BP] + [APMA]) on the diameter and size distribution of the formed polyMA-Glu-BP NPs.

Effepan class="Chemical">ct of the weight % ratio [pan class="Chemical">TTEGDA]/([MA-Glu-BP] + [APMA]) on the diameter and size distribution of the formed polyMA-Glu-BP NPs.

Each sampn>le had a combined monomer concentration of 2.5% (w/v). The ratio between the monomers is represented by the expression [TTEGDA]/([MA-Glu-BP] + [APMA]). Figure demonstrates that when the ratio of [TTEGDA]/([MA-Glu-BP] + [APMA]) is raised from 1 to 1.2%, the diameter decreases from 197 ± 21 to 163 ± 11 nm, respectively. At 2.5%, there is a further decrease in the diameter to 136 ± 12 nm. At 3.4 and 5%, similar diameters were measured, which are 129 ± 7 and 130 ± 6 nm, respectively. The decrease in the NP diameter and size distribution with increase in the cross-linker concentration could be explained by the difficulties in the growth of the highly cross-linked NPs relative to the less cross-linked nuclei due to the monomer swelling. The swelling process in water is generally more difficult with higher particle cross-linkage, as already indicated in the literature.[44−46]

Effect of Mw and Concentration of the Stabilizer Polyvinylpyrrolidone

Figure illustrates the effect of the molecular weight (40k and 360k g/mol) and concentration of the stabilizer polyvinylpyrrolidone (PVP) on the hydrodynamic diameter and size distribution of the polyMA-Glu-BP NPs. At a concentration of 0.05%, the particle diameter obtained was 531 ± 43 nm for PVP of 40k molecular weight and 350 ± 14 nm for PVP of 360k molecular weight. At a concentration of 0.125%, both stabilizers decrease the diameter of the particles to 473 ± 57 nm for 40k and 301 ± 10 nm for 360k. At 0.25%, both stabilizers continued to reduce the diameter of the polyMA-Glu-BP NPs, to a diameter of 462 ± 50 nm for 40k and 226 ± 7 nm for 360k. At 0.5%, the diameter further declined to 431 ± 44 nm for 40k and 183 ± 11 nm for 360k. A moderate decrease in the particle diameter was obtained for PVP of 360k molecular weight; at 1 and 2% stabilizer concentration, the diameters were 175 ± 9 and 160 ± 15 nm, respectively. However, PVP of 40k molecular weight at 1% concentration exhibited a sharp decrease in the particle diameter, resulting in a diameter of 278 ± 47 nm, and reached a plateau at 2% concentration with a diameter of 272 ± 48 nm. Both stabilizers demonstrate a similar trend of inverse relationship between particle diameter and stabilizer concentration. These results can be attributed to the adsorption of the stabilizer on the surface of the particles, limiting the growth. PVP of 360k molecular weight results in particles with a smaller diameter and a smaller size distribution than 40k at similar concentrations. This difference between both stabilizers could be explained by the greater absorption of the stabilizer with a higher molecular weight on the surface of the particles, therefore restricting the growth of the particles.[47−50] The same reason may also explain the significantly lower size distribution of the NPs obtained with PVP of 360k molecular weight relative to that obtained with PVP of 40k molecular weight, as shown in Figure .

Figure 8

Effect of the molecular weight (40k and 360k) and concentration of the stabilizer PVP on the hydrodynamic size and size distribution of the formed polyMA-Glu-BP NPs.

Effepan class="Chemical">ct of the molepan class="Chemical">cular weight (40k and 360k) and concentration of the stabilizer PVP on the hydrodynamic size and size distribution of the formed polyMA-Glu-BP NPs.

Effect of the Initiator Concentration

Figure represents the relationship between the initiator [potassium persulfate (PPS)] concentration and the hydrodynamic diameter and size distribution of the particles. By increasing the initiator concentration from 1 to 2%, the diameter of the particles is slightly reduced from 421 ± 14 to 358 ± 11 nm, respectively. At 3%, there is a dramatic decrease in the diameter to 187 ± 9 nm, followed by a mild decrease at 4% to 160 ± 13 nm. At the following concentrations, the diameters remain constant: 160 ± 6 nm for 8% and 163 ± 9 nm for 16%. These results can be attributed to the fact that the higher the concentration of initiator, the greater the number of polymerization sites; hence more particles of smaller diameter are formed, as reported in the literarure.[44,48,51]

Figure 9

Effect of the concentration of PPS on the diameter of formed particles.

Effepan class="Chemical">ct of the concentration of PPS on the diameter of formed particles.

Effect of Temperature

Figure exhibits the effect of the reaction temperature on the hydrodynamic diameter and size distribution of the formed particles. The figure demonstrates that by raising the temperature from 70 to 75 °C, the particle diameter decreases from 192 ± 10 to 180 ± 10 nm, respectively. At 80 °C, the particle diameter further decreases to 160 ± 6 nm and remains similar at 85 °C (160 ± 11 nm). These findings could be explained by the fact that the increase in temperature causes an increase in the decomposition rate of the initiator PPS, which leads to an increase in the number of polymerization sites created. Consequently, more particles are formed but of smaller diameter.[41]

Figure 10

Effect of the reaction temperature on the diameter of the formed particles.

Effepan class="Chemical">ct of the reapan class="Chemical">ction temperature on the diameter of the formed particles.

Calcium Affinity Test Using Coral

Coral skeletons contain large amounts of calcium carbonate (CaCO3),[52,53] making their fragments a useful tool for in vitro calcium affinity testing. Therefore, the adsorption of the near-infrared (NIR) fluorescent polyMA-Glu-BP NPs compared with NIR fluorescent control NPs (containing methacryloylglutamine monomer instead of the MA-Glu-BP monomer) toward coral was demonstrated using a NIR fluorescence microscope (Figure ). Corals treated with 0.5 mg/mL polyMA-Glu-BP NPs (Figure F) exhibit a prominent fluorescence compared to corals treated with 0.5 mg/mL control NPs (Figure D), which do not exhibit any noticeable fluorescence. The fluorescence intensities of the corals treated with polyMA-Glu-BP NPs and control NPs were compared using ImageJ software. The results demonstrated that the corals treated with polyMA-Glu-BP NPs exhibited an 11 times greater fluorescence intensity than the control NPs.

Figure 11

Fluorescence microscopy images: bright-field images of the corals treated with water (A), 0.5 mg/mL NIR fluorescent control NPs (C), and 0.5 mg/mL NIR fluorescent polyMA-Glu-BP NPs (E) and NIR images of the corals treated with water (B), 0.5 mg/mL NIR fluorescent control NPs (D), and 0.5 mg/mL NIR fluorescent polyMA-Glu-BP NPs (F).

In addition, the results of high-resolution scanning electron microscopy (HR-SEM) analysis of the corals treated with water, control NPs (0.5 mg/mL), and polyMA-Glu-BP NPs (0.5 mg/mL) are presented in Figure . There is a distinct difference between the surfaces of the various treated corals. The surface of the corals treated with water (Figure A) and control NPs (Figure B) appear similar and are composed of spikey panels. In contrast, the corals treated with the polyMA-Glu-BP NPs (Figure C) exhibit a smooth surface, indicating that the surface of the coral is coated by the NPs. These results can be attributed to the affinity of the BP groups to the calcium ions in the coral in comparison to the control NPs. These findings confirm that the polyMA-Glu-BP NPs have strong affinity toward calcium in comparison to the control NPs.

Figure 12

HR-SEM analysis of the corals treated with water (A), 0.5 mg/mL control NPs (B), and 0.5 mg/mL polyMA-Glu-BP NP (C) aqueous dispersion, as described in the Experimental Section.

HR-SEM analysis of the corals treated with pan class="Chemical">water (A), 0.5 mg/mL control NPs (B), and 0.5 mg/mL polyMA-Glu-BP NP (C) aqueous dispersion, as described in the Experimental Section.

Toxicity of the PolyMA-Glu-BP NPs

pan class="Disease">Cytotoxicity and pan class="Disease">toxicity assays [lactate dehydrogenase (LDH) and 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) tests] were performed on J774A1, hFOB 1.19, and RAW 264.7 human cell lines. The results of LDH and XTT tests of all three types of cells are summarized in Figure . The cells were treated for 48 and 72 h with a serial diluted concentration of the polyMA-Glu-BP NP aqueous dispersion (1–1000 μg/mL). The LDH results obtained (Figure A) showed negligible cytotoxicity after 48 h of incubation for all three cell lines. Similar results were demonstrated following 72 h of treatment with J774A1 and hFOB 1.19 cell lines; RAW 264.7 exhibited slight cytotoxicity (Figure B). The XTT results in Figure C,D for 48 and 72 h of treatment, respectively, exhibit that the viability of the cells was unharmed. Therefore, it can be concluded that the NPs have negligible toxicity under these conditions.

Figure 13

Toxicity tests of the polyMA-Glu-BP NPs on J774A1, hFOB 1.19, and RAW 264.7 cells: (A) LDH test after 48 h, (B) LDH test after 72 h, (C) XTT test after 48 h, and (D) XTT test after 72 h.

pan class="Disease">Toxicity tests of the pan class="Chemical">polyMA-Glu-BP NPs on J774A1, hFOB 1.19, and RAW 264.7 cells: (A) LDH test after 48 h, (B) LDH test after 72 h, (C) XTT test after 48 h, and (D) XTT test after 72 h.

Cellular Uptake of Cy7-polyMA-Glu-BP NPs by J774A1 Macrophages

To perform kinetic cellular uptake studies of the polyMA-Glu-BP NPs, the fluorescent dye cyanine7 (Cy7) was conjugated to the free amine of the polyMA-Glu-BP NP surface (as described in the Experimental Section). Kinetic cellar uptake of the Cy7-polyMA-Glu-BP NPs (0.1 mg/mL) by J774A1 macrophage cells was performed (Figure ). The results summarized in Table clearly demonstrate that the uptake of the NPs by the J774A1 macrophage cells occurs in large quantity after just 1 h of treatment, indicated by the Cy7 fluorescence (99.55 ± 0.07%). The fluorescence values remain similar for the following hours: 99.9 ± 0% after 5 h and 99.95 ± 0.07% after 24 h.

Figure 14

Kinetic cellar uptake of the Cy7-polyMA-Glu-BP NPs in J774A1 macrophage cells at various times.

Table 1

Kinetic Cellar Uptake of the Cy7-polyMA-Glu-BP NPs in J774A1 Macrophage Cells at Various Timesa

treatment (h)	Cy7 positive cells (%)
1	99.5 ± 0.07
5	99.9 ± 0
24	99.9 ± 0.07
untreated cells	0.35 ± 0.03

Antibacterial activity of the polyMA-Glu-BP NPs.

Kinetipan class="Chemical">c cellar uptake of the Cy7-polyMA-Glu-BP NPs in J774A1 macrophage cells at various times.

Antibapan class="Chemical">cterial apan class="Chemical">ctivity of the polyMA-Glu-BP NPs.

The antibacterial activity of the polyMA-Glu-BP NPs was evaluated as described in the Experimental Section, by determining their minimum bactericidal concentration (MBC) using Escherichia coli and Pseudomonas aeruginosa, two common bacterial pathogens representing Gram-negative bacteria, and Listeria and Staphylococcus aureus bacteria, which represent Gram-positive bacteria. The bacteria were exposed to a serial diluted concentration of polyMA-Glu-BP NPs in an aqueous dispersion, and the MBC was found to be 0.93 and 7.5 mg/mL for P. aeruginosa and E. coli, respectively. The MBC for Listeria was found to be 1.87 mg/mL (Table ). Notably, neither of the tested concentrations demonstrated total kill of S. aureus. Nevertheless, at the range 7.5–0.23 mg/mL, there was a partial killing with 7.5 mg/mL, leading to a reduction of 2.5 logs in the viability of the bacteria.

Table 2

MBC of the PolyMA-Glu-BP NPs

bacterial type	MBC (mg/mL)
E. coli	7.5
P. aeruginosa	0.93
Listeria	1.87

Summary and Conclusions

In this work, the synthesis of the vinylic monomer MA-Glu-BP and the polyMA-Glu-BP NPs has been reported. To obtain an optimal particle, the influence of the following parameters on the diameter of the polyMA-Glu-BP NPs was studied: total monomer concentration, cross-linker concentration, stabilizer Mw and concentration, initiator concentration, reaction temperature, and reaction time. The optimal polyMA-Glu-BP NPs obtained have a dry diameter of 61.2 ± 6 nm and a hydrodynamic diameter of 163.2 ± 7 nm.

The surfapan class="Chemical">ce pan class="Chemical">BP functional groups have a high affinity to calcium ions. This property was demonstrated by treating coral fragments with polyMA-Glu-BP NPs and compared to those of double-distilled water (DDW) and control NPs. The polyMA-Glu-BP NPs exhibited a high affinity to the coral, which was 11 times greater than that of the control NPs.

A cytotoxicity study of the polyMA-Glu-BP NPs exhibited no cytotoxic effect under the experimental conditions on J774A1, hFOB 1.19, and RAW 264.7 cells. On the other hand, the polyMA-Glu-BP NPs exhibited toxicity toward the Gram-negative bacteria E. coli and P. aeruginosa and the Gram-positive bacteria Listeria. These findings may suggest that the polyMA-Glu-BP NPs have a potential to be safely used in humans against bacterial infections. This could be explained by the fact that calcium is highly abundant in the bacterial cell wall structure; therefore, chelating agents can easily disrupt the calcium homeostasis of the bacteria. However, the mammalian cell membrane contains much less calcium. Therefore, chelating agents cannot significantly disrupt the calcium homeostasis of the mammalian cells.[4,5] Therefore, further research is needed. In the future, additional studies should be performed to understand the mechanism of toxicity of these NPs toward different bacteria. Furthermore, because of the presence of the surface amine, other antibacterial drugs can be conjugated to the NPs in an attempt to increase the toxic effect toward bacteria.

Experimental Section

Materials

The following analytical-grade chemicals were purchased from commercial sources and used without further purification: glutamic acid, acetic anhydride, phthalic anhydride, TTEGDA, PPS, PVP (Mw 40k and Mw 360k), sodium hydroxide (1 N), hydrochloric acid (1 N), anhydrous dichloromethane, anhydrous tetrahydrofuran (THF), anhydrous methanol, anhydrous dimethyl sulfoxide (DMSO), O-[(N-succinimidyl)succinyl-aminoethyl-O′-methylpolyethylene glycol (PEG-NHS, Mw 750), methacryloyl chloride, tris(trimethylsilyl)phosphite, and glutamine from Sigma (Rehovot, Israel); APMA from Polysciences, Inc. (Warrington, PA); dialysis membrane (1000k—16 mm), sodium carbonate, and sodium bicarbonate from Bio-Lab Ltd. (Jerusalem, Israel); cyanine7 N-hydroxysuccinimide (Cy7-NHS) ester from Lumiprobe Corporation (Florida, USA); Dulbecco’s phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), glutamine, and penicillin/streptomycin from Biological Industries (Beit Haemek, Israel); human osteosarcoma cell line Saos-2 and human colon carcinoma cell line SW620 from American Type Culture Collection (Manassas, VA); and Matrigel from Sigma (Germany). DDW was purified by passing deionized DDW through an Elgastat Spectrum reverse osmosis system (ELGA Ltd., High Wycombe, UK). Coral scaffold was received as a gift from Boneus Ltd. (Haifa, Israel).

Synthesis of the Vinylic Monomer MA-Glu-BP

pan class="Chemical">1H NMR, pan class="Chemical">13C NMR, and 31P NMR spectral data were obtained with the following Bruker NMR spectrometers: AVANCE II 300 MHz, AVANCE I 400 MHz, and DMX 600 MHz. Deuterated solvents were purchased from Tzamal D-Chem Laboratories Ltd. by the Nuclear Magnetic Resonance Facility of Bar Ilan University.

Fourier transform infrpan class="Chemical">ared (FTIR) analysis was performed with a Bruker Platinum-FTIR QuickSnap sampling module A220/D-01. The analysis was performed with 13 mm KBr pellets composed of 2 mg of the detected material (MSF or PMSF) and 198 mg of KBr. The pellets were scanned over 48 scans at a 4 cm–1 resolution.

Elemental analysis was performed with a PerkinElmer 2400 series II analyzer, by the analytipan class="Chemical">cal laboratories of the Hebrew University, Jerusalem.

Low-resolution mass spepan class="Chemical">ctra were obtained on a Micromass Q-Tof microspectrometer in the electrospray mode.

Synthesis and Characterization of N-Phthaloyl Glutamic Acid

N-pan class="Chemical">Phthaloyl pan class="Chemical">glutamic acid was synthesized similar to the procedure described in the literature.[32] In brief, acetic acid (34 mL) was added to a 500 mL round-bottom flask containing phthalic anhydride (12.88 g, 0.087 mol) and l-glutamic acid (17 g, 0.11 mol), resulting in a turbid solution. The solution was heated at 145 °C for 1.5 h, giving a clear colorless solution. The solution was allowed to cool to room temperature (rt) and then evaporated to give a colorless oil. DDW (334 mL) was then added to the oil and heated to 100 °C, resulting in a clear colorless solution. The solution was then allowed to cool to rt, 32% HCl (6 mL) was added, and the mixture was refrigerated overnight. The obtained solid was filtered and dried to yield the desired product (10.7 g, 44.4% yield).

pan class="Chemical">1H NMR (pan class="Chemical">DMSO-d6) 300 MHz: 2.21–2.39 (m, 4H, HO2C–CH–CH), 4.78–4.83 (m, 1H, N–CH–CO2H), 7.85–7.92 (m, 4H, Ar–H).

TOF MS+ (m/z): [M + Na]+ pan class="Chemical">calpan class="Chemical">cd for C13H11NO6Na+, 300.05; found, 300.

Synthesis and Characterization of N-Phthaloyl Glutamic Anhydride

pan class="Chemical">N-Phthaloyl glutamic anhydride was synthesized similpan class="Chemical">ar to the procedure described in the literature.[32] In brief, a suspension of N-phthaloyl glutamic acid (10 g, 0.036 mol) in acetic anhydride (40 mL) was heated at 100 °C for 2 h to give a clear colorless solution, then cooled to rt, and evaporated, yielding the desired compound, a white pinkish solid (8.6 g, 91.6% yield).

pan class="Chemical">1H NMR (pan class="Chemical">DMSO-d6) 300 MHz: 2.12–2.18 (m, 1H, N–CH–CH2–CH–CO2−), 2.56–2.71 (m, 1H, N–CH–CH–CH2–CO2−), 2.96–3.02 (m, 1H, N–CH–CH2–CH–CO2−), 3.08–3.20 (m, 1H, N–CH–CH2–CH–CO2−), 5.45–5.52 (m, 1H, N–CH–CO2−), 7.87–7.95 (m, 4H, Ar–H).

TOF MS+ (m/z): [M + H]+ pan class="Chemical">calpan class="Chemical">cd for C13H10NO5+, 260.06; found, 260.

Synthesis and Characterization of N-Pht-Glu-BP

N-pan class="Chemical">Phthaloyl pan class="Chemical">glutamic anhydride (8 g, 0.031 mol) was dissolved in 200 mL of dry THF under a nitrogen atmosphere to give a clear off-white solution. Tris(trimethylsilyl)phosphite (20 g,0.07 mol) was then added to the solution and stirred overnight. The obtained clear colorless solution was evaporated, resulting in a clear pinkish oil, which was then dissolved in methanol (150 mL) and stirred for 2 h to give a clear colorless solution. The methanol solution was evaporated, and the resulting orange oil was washed with diethyl ether (700 mL) and dried to yield the desired compound as a white solid (10.8 g, 83.3% yield).[33]

pan class="Chemical">1H NMR (pan class="Chemical">D2O) 600 MHz: 1.86–2.05 (m, 2H, P–C–CH2), 2.36–2.52 (m, 2H, P–C–CH2–CH2) 4.85–4.89 (m, 1H, N–CH–CO2H), 7.75–7.83 (m, 4H, Ar–H). 13C NMR (D2O) 600 MHz: 24.26 (t, 3Jcp = 7 Hz, P–C–CH2–CH2), 31.08 (s, P–C–CH2), 53.48 (s, N–CH–CO2H), 73.89 (t, 1Jcp = 138 Hz, P–C–P), 124.32 (s, CO–Ar), 131.66 (s, CO–Ar), 135.58 (s, CO–Ar), 170.43 (s, Ar–CO–N), 173.99 (s, CO2H). 31P NMR (D2O) 400 MHz: 19.13 (s).

TOF MS– (m/z): [M – H]− pan class="Chemical">calpan class="Chemical">cd for C13H14NO11P2–, 422.2; found, 422.

FTIR (KBr): 3429 (pan class="Chemical">COOH), 1773 (pan class="Chemical">COOH), 1711 (Ar–C=O), 1396 (CO2–), 1174 (P=O), 1070 (P–C–OH), 930 (COOH dimer), 722 (P–C), 532 (O–P–O).

Elemental analysis: Elemental analysis pan class="Chemical">calpan class="Chemical">cd for C13H15NO11P2·2H2O (459.03): C, 34.00; H, 4.17; N, 3.05; P, 13.49. Found: C, 33.10; H, 3.32; N, 2.98; P, 13.29.

Depan class="Chemical">compn>oses at 290 °pan class="Chemical">C.

Synthesis and Characterization of γ-Glu-BP

pan class="Chemical">N-Pht-Glu-BP (10.8 g, 0.036 mol) was dissolved in 6 M pan class="Chemical">HCl solution (180 mL), and the clear colorless solution was refluxed overnight. The solution was cooled to rt and then refrigerated for 4 h. The resulting white crystals of phthalic acid were filtered, and the clear colorless filtrate was evaporated to yield the desired compound as a white solid (5.9 g, 98.7% yield).[34]

pan class="Chemical">1H NMR (pan class="Chemical">D2O) 400 MHz: 2.03–2.34 (m, 4H, P–C–CH–CH), 4.06–4.09 (m, 1H, N–CH–CO2H). 13C NMR (D2O) 400 MHz: 25.66 (t, 3Jcp = 9 Hz, P–C–CH2–CH2), 29.76 (s, P–C–CH2), 54.01 (s, NH2–CH–CO2H), 73.53 (t, 1Jcp = 183 Hz, P–C–P), 173.04 (s, CO2H). 31P NMR (D2O) 400 MHz: 18.74 (s).

TOF MS+ (m/z): [M + H]+ pan class="Chemical">calpan class="Chemical">cd for C5H14NO9P2+, 294.01; found, 294.

FTIR (KBr): 3170 (pan class="Chemical">COOH), 1732 (pan class="Chemical">COOH), 1632 (H–N), 1528 (CO2–), 1162 (P=O), 1064 (P–C–OH), 919 (COOH dimer), 529 (O–P–O).

Elemental analysis: elemental analysis pan class="Chemical">calpan class="Chemical">cd for C5H13NO9P2·2H2O (329.03): C, 18.25; H, 5.21; N, 4.26; P, 18.82. Found: C, 15.56; H, 4.52; N, 3.79; P, 18.92.

Depan class="Chemical">compn>oses at 290 °pan class="Chemical">C.

Synthesis and Characterization of the Monomer MA-Glu-BP

pan class="Chemical">Methacryloyl chloride (1 mL, 0.01 mol) was added dropn>wise to a solution of γ-pan class="Chemical">Glu-BP (2 g, 0.007 mol) and sodium hydroxide (2 g, 0.05 mol) in DDW (20 mL) at 0 °C. The resulting solution was stirred at 0 °C for 1.5 h and an additional 3.5 h at rt, and a clear colorless solution and white emulsions were obtained. After removing the white emulsions, ethanol (250 mL) was added to the clear solution, resulting in a white precipitant, which was collected by Buchner filtration and washed with additional ethanol (850 mL), yielding the desired product as a salt (2.5 g, 76.0% yield).[35,36]

pan class="Chemical">1H NMR (pan class="Chemical">D2O) 400 MHz: 1.84–2.21 (m, 4H, P–C–CH–CH), 1.96 (s, 3H, CH2=C–CH), 4.09–4.12 (m, 1H, N–CH–CO2H), 5.47 (s, 1H, cis CH=C–CH3), 5.77 (s, 1H, trans CH=CH3). 13C NMR (D2O) 400 MHz: 18.48 (s, CH2=C–CH3), 27.39–27.52 (t, 3JCP = 24 Hz, P–C–CH2–CH2), 32.75 (s, P–C–CH2), 57.53 (s, NH–CH–CO2H), 74.24–76.83 (t, 1JCP = 130 Hz, P–C–P), 121.58 (s, CH2=C–CH3), 139.87 (s, CH2=C–CH3), 172.44 (s, CO–NH), 180.53 (s, CO2H). 31P NMR (D2O) 400 MHz: 18.78–19.38 (q, JPP = 19.96 Hz).

TOF MS– (m/z): [M – H]− pan class="Chemical">calpan class="Chemical">cd for C9H16NO10P2–, 360.03; found, 360.

FTIR (KBr): 3406 (H–N), 2233 (pan class="Chemical">C=pan class="Chemical">C), 1655 (CO–N–H), 1594 (CO2), 1455 (CH2), 1408 (CH3), 1222 (P=O), 1107 (P–C–OH), 1003 (COOH dimer), 957 (C=CH2), 557 (O–P–O).

Elemental analysis: elemental analysis pan class="Chemical">calpan class="Chemical">cd for C9H12NO10P2Na5·4H2O (542.98): C, 19.90; H, 3.71; N, 2.58; P, 11.41. Found: C, 18.03; H, 3.67; N, 1.35; P, 10.77.

Depan class="Chemical">compn>oses at 290 °pan class="Chemical">C.

Synthesis of the Cross-Linked PolyMA-Glu-BP NPs

pan class="Chemical">MA-Glu-BP (17.5 mg), 5 mg of pan class="Chemical">APMA, and 27.5 mg of the cross-linker monomer TTEGDA (a total monomer concentration of 2.5% w/v) were added to a vial containing 4 mg of the initiator PPS (4 w/w %) and 20 mg of PVP of 360k molecular weight (1 w/v %) as a stabilizer dissolved in 2 mL of 0.035 M HCl (aq). The vial containing the mixture was purged with N2 to exclude air and then shaken at 80 °C overnight. The obtained polyMA-Glu-BP NPs were washed of excess reagents by extensive dialysis cycles (cut-off of 1 000 000k) with DDW, giving particles with a hydrodynamic diameter of 163.2 ± 7 nm and a dry diameter of 61.2 ± 6 nm.

The Npan class="Chemical">P formation yield, 33.12%, was pan class="Chemical">calculated by the following expression:where Wparticles is the weight of the dried particles and Wtotal monomers is the initial weight of the three polymerized monomers.

The pan class="Chemical">conpan class="Chemical">centration of the BP groups of the particles, as calculated from the elemental analysis, was 0.2 mmol/g particles.

Elemental analysis: pan class="Chemical">C, 52.84% (pan class="Chemical">calcd 50.32%); H, 7.02% (calcd 6.95%); N, 1.31% (calcd 4.08%); P, 0.59% (calcd 4%).

Characterization of the PolyMA-Glu-BP NPs

The dry diameter and size distribution of the NPs were measured with TEM. TEM images were obtained with an FEI Tecnai C2 BioTWIN electron microscope with a 120 kV accelerating voltage. Samples for TEM were prepared by placing a drop of diluted sample on a 400 mesh carbon-coated copper grid previously exposed to plasma for 10 s. The average particle diameter and size distribution were determined by the measurement of the diameter of more than 200 particles. The hydrodynamic diameter and size distribution of the particles dispersed in DDW were measured at rt with a particle analyzer, model NANOPHOX (SympatecGmbH, Germany).

Elepan class="Chemical">ctrokinetipan class="Chemical">c properties (ζ-potential) of the formed particles were measured using a titration method, from pH 2.5 to 10.9 with 0.1 M HCl and 0.1 M NaOH. The measurements were measured at a constant ionic strength of 0.1 M. The ζ-potential of the formed particles was measured by a ζ-potential analyzer model Zetasizer 3000 HSa (Malvern Instruments, UK).

Elemental analysis was performed with a PerkinElmer 2400 series II analyzer, by the analytipan class="Chemical">cal laboratories of the Hebrew University, Jerusalem. pan class="Chemical">Phosphorus content was determined using the oxygen flask combustion method followed by ion chromatography analysis using a Dionex IC system.

Synthesis of the Cy7-Conjugated BP NPs

Cy7-conjugated polyMA-Glu-BP NPs were prepared by the reaction of the primary amino groups (belonging to the APMA monomeric units) on the polyMA-Glu-BP NP surface with Cy7-NHS ester. Briefly, Cy7-NHS ester (0.1 mg) was dissolved in anhydrous DMSO and added to 5 mL of the polyMA-Glu-BP NP dispersion in 0.1 M BB (2 mg/mL), and the reaction mixture was stirred for 1 h at rt. The residual amine groups were then blocked by the addition of 5 mg of PEG-NHS (Mw 750) to the NIR fluorescent polyMA-Glu-BP NP aqueous dispersion and stirred at rt for an additional 1 h. The obtained NIR fluorescent conjugated polyMA-Glu-BP NPs were then washed of excess reagents by extensive dialysis in DDW (a cutoff of 1 000 000k). The excitation maxima of the dye shift from 746 to 768 nm because of the conjugation to the NPs, and the emission maxima shift from 766 to 789 nm.

Synthesis and Characterization of the Monomer Methacryloylglutamine

Methacryloyl chloride (5.63 mL, 0.058 mol) was added dropwise to a solution of glutamine (2.1 g, 0.014 mol) and sodium hydroxide (3.2 g, 0.08 mol) in DDW (40 mL) at 0 °C. The resulting solution was stirred at 0 °C for 1.5 h and an additional 3.5 h at rt, giving a clear colorless solution and a white soft solid, which were separated by filtration. The aqueous filtrate was acidified to pH 1 with HCl 32% until a white precipitant appeared. The precipitant was filtered off, and remaining aqueous solution was washed with EtOAc (6 × 50 mL). The water was evaporated to give the desired product (2.76 g, 92.9% yield).[35,36]

pan class="Chemical">1H NMR (pan class="Chemical">D2O) 300 MHz: 1.99 (s, 3H, CH2=C–CH), 2.05–2.34 (m, 2H, H2N–CO–CH2–CH−), 2.43–2.48 (t, 2H, H2N–CO–CH–CH2−), 4.43–4.48 (quart, 1H, N–CH–CO2H), 5.56 (s, 1H, cis CH=C–CH3), 5.79 (s, 1H, trans CH=CH3).

pan class="Chemical">13C NMR (pan class="Chemical">D2O) 400 MHz: 18.40 (s, CH2=C–CH3), 27.13 (s, H2N–CO–CH2–CH2−), 32.75 (s, H2N–CO–CH2–CH2−), 53.68 (s, NH–CH–CO2H), 122.43 (s, CH2=C–CH3), 139.45 (s, CH2=C–CH3), 172.65 (s, CO–NH), 176.39 (s, H2N–CO–CH2–CH2−), 178.92 (s, CO2H).

Synthesis of the Control NPs

pan class="Chemical">Methacryloylglutamine (70 mg), 20 mg of pan class="Chemical">APMA, and 110 mg of the cross-linker monomer TTEGDA (a total monomer concentration of 2.5 w/v %) were added to a vial containing 16 mg of the initiator PPS (4 w/w %) and 80 mg of PVP of 360k molecular weight (1 w/v %) as a stabilizer dissolved in 8 mL of 0.035 M HCl (aq). The vial containing the mixture was purged with N2 to exclude air and then shaken at 80 °C overnight. The obtained NPs were washed of excess reagents by extensive dialysis cycles (a cutoff of 1 000 000k) with DDW, giving particles with a hydrodynamic diameter of 149.1 ± 36 nm.

The Npan class="Chemical">P formation yield, 65.92%, was pan class="Chemical">calculated by the following expression:where Wparticles is the weight of the dried particles and Wtotal monomers is the initial weight of the three polymerized monomers.

Synthesis of the NIR Fluorescent Conjugated Control NPs

Cy7-conjugated polyMA-Glu-BP NPs were prepared by the reaction of the primary amino groups (belonging to the APMA monomeric units) on the polyMA-Glu-BP NP surface with Cy7-NHS ester. Briefly, Cy7-NHS ester (0.1 mg) was dissolved in anhydrous DMSO and added to 5 mL of the control NP dispersion in 0.1 M BB (2 mg/mL), and the reaction mixture was stirred for 1 h at rt. The residual amine groups were then blocked by the addition of 5 mg of PEG-NHS (Mw 750) to the NIR fluorescent control NP aqueous dispersion and stirred at rt for an additional 1 h. The obtained NIR fluorescent conjugated control NPs were then washed of excess reagents by extensive dialysis in DDW (a cutoff of 1 000 000k).

Calcium Affinity Test of the PolyMA-Glu-BP NPs

The affinity of the polyMA-Glu-BP NPs to calcium ions was evaluated by incubating coral fragments (5.55 mg) with NIR fluorescent polyMA-Glu-BP NP dispersion (200 μL, 0.5 mg/mL) for 2 h. The solution was removed, and the coral was washed three times with 1 mL of DDW. As a reference, coral fragments were treated similarly with the NIR fluorescent control NP dispersion (200 μL, 0.5 mg/mL) and DDW. The coral fragments were evaluated by a fluorescent microscope Olympus BX 60 Qimaging EXi Blue QCcapture X-64. In addition, the coral fragments were then attached to a stab with carbon glue tape, coated with carbon in vacuum, and analyzed with a high-resolution scanning electron microscope (FEI, Magellan 400L).

In Vitro Cytotoxicity of the PolyMA-Glu-BP NPs

In vitro cytotoxicity of the polyMA-Glu-BP NPs was tested by using RAW 264.7 (mice macrophages; Abelson murine leukemia virus-transformed), J774A.1 (mice macrophages; reticulum cell sarcoma), and hFOB 1.19 (human osteoblast; SV40 large T antigen-transfected) cell lines. The cells are adherent to the culture dishes. RAW 264.7 and J774A.1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with heat-inactivated FBS (10%), penicillin (100 IU/mL), streptomycin (100 μg/mL), and l-glutamine (2 mM). hFOB 1.19 cells were maintained in 1:1 mixture of Ham’s F12 medium and Dulbecco’s modified Eagle’s medium (without phenol red), with heat-inactivated FBS (10%), penicillin (100 IU/mL), streptomycin (100 μg/mL), 2 mM l-glutamine, and 0.3 mg/mL G418. Cells were screened to ensure that they remained mycoplasma-free using a mycoplasma detection kit.[54]

Cell cytotoxicity was assessed by measuring the release of cytoplasmic LDH into cell culture supernatants. LDH activity was assayed using the cytotoxicity detection kit according to the manufacturer’s instructions.[55] Briefly, cells (5 × 103 cells per well) were seeded and grown for 48 h in 96-well plates before treatment with the polyMA-Glu-BP NPs. Cell cultures that were not exposed to the NPs were included in all assays as negative controls. Cell cultures that were treated with cell lysis solution were used as positive controls.

The pan class="Chemical">polyMA-Glu-BP NPs were freshly dispersed in pan class="Chemical">PBS (1.25 and 2.5 mg/mL) and then added to the 95% confluent cell culture in culture medium. The cell cultures were further incubated at 37 °C in a humidified 5% CO2 incubator and then checked for cellular cytotoxicity after 48 h of incubation. The percentage of cell cytotoxicity was calculated using the formula shown in the manufacturer’s protocol.[55] All samples were tested in tetraplicates.

Cell Proliferation Analysis

XTT analysis[56] was performed according to the manufacturer’s instructions (Biological Industries). In brief, RAW 264.7 (mice macrophages; Abelson murine leukemia virus-transformed), J774A.1 (mice macrophages; reticulum cell sarcoma), and hFOB 1.19 (human osteoblast; SV40 large T antigen-transfected) cells were seeded onto 96-well plates and incubated for 48 h at 37 °C in 5% CO2. The polyMA-Glu-BP NPs were freshly dispersed in PBS (1.25 and 2.5 mg/mL) and then added to the 95% confluent cell culture in culture medium. The cell cultures were further incubated at 37 °C in a humidified 5% CO2 incubator and then checked for cellular viability after 48 h of incubation. The percentage of cell viability was calculated using the formula shown in the manufacturer’s protocol.[56] All samples were tested in tetraplicates.

Flow Cytometry Analyses of NP Uptake

To study the effect of the incubation time on the uptake of polyMA-Glu-BP NPs, J774A.1 cells were incubated with Cy7-conjugated BP NPs (at 0.3 mg/mL) for 1, 5, and 24 h. Cells were then washed twice with fresh medium and collected in the dark. The uptake of the Cy7-conjugated NPs within cells was evaluated by FACSAria III (BD) cell sorting. To maximize the cell viability and minimize the mechanical perturbations, the flow rate was set to 1.1 (minimum). A minimum of 10 000 cells were analyzed for each histogram generated. Gate SSC/FSC was used to exclude fragments and aggregates from the cell count. For Cy7 analysis, a 633 nm excitation laser was used with a filter. Data were processed by FlowJo v7.6.4.[57]

Bacterial Cultures and Growth Conditions

Bapan class="Chemical">cterial pan class="Chemical">cultures and growth conditions were similar to those reported in the literature.[25,29]E. coli ATCC 25922, S. aureus ATCC 29213, and P. aeruginosa PAO1 were grown overnight at 37 °C under agitation (250 rpm) in Mueller Hinton (MH, Difco), and Listeria innocua ATCC 33090 was grown in Brain Heart (BH, Difco) growth media.

Antibacterial Activity Assay of the PolyMA-Glu-BP NPs

The antibacterial activity of the polyMA-Glu-BP NPs was evaluated by determining the MBC values for all bacterial strains tested. The stock dispersion of the NPs was diluted in two-fold serial dilutions ranging from a concentration of 7.5 to 0.058 mg/mL in saline solution in a 96-well plate (Greiner Bio-One). Each well contained 105 colony-forming units (CFUs)/mL of each bacterial strain. Bacteria treated with DDW served as a negative control. The following day, 10-fold serial dilutions were carried out and the bacterial cells were plated on LB agar plates, followed by their incubation at 37 °C for 20 h. Cell growth was monitored and determined by viable cell count and expressed as CFUs. All experiments were conducted in duplicates at least three independent times.