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

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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.
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Engineering of a New Bisphosphonate Monomer and Nanoparticles
of Narrow Size Distribution for Antibacterial Applications
Nimrod Tal,
Safra Rudnick-Glick,
Igor Grinberg,
Michal Natan,
Ehud Banin,
and Shlomo Margel*
,
The Institute of Nanotechnology and Advanced Materials, Department of Chemistry, and
The Mina and Everard Goodman Faculty
of Life Sciences, The Institute for Advanced Materials and Nanotechnology, Bar-Ilan University, Ramat-Gan 52900, Israel
ABSTRACT: 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 anity to calcium ions and
are eective 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 bi-
sphosphonate) and polyMA-Glu-BP nanoparticles (NPs) for
antibacterial applications. polyMA-Glu-BP NPs were synthe-
sized 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
uorescent 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 anity of the near-infrared
uorescent 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.
1. INTRODUCTION
Bacteria play a major role in 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 eective 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
912
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 POP
repeating unit. Endogenous pyrophosphate is the smallest
polyphosphate (Figure 1).
15
Bisphosphonates (BPs) are a group of stable organic
analogues of the pyrophosphate (Figure 1). BPs have high
anity to calcium and are known as eective calcium
chelators.
1624
Considering the similarity of BPs to poly-
phosphates, they could be used as antibacterial agents.
25
Nanoparticles (NPs) are spherical macromolecules with a
diameter of 1100 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
Received: October 31, 2017
Accepted: January 15, 2018
Published: February 2, 2018
Figure 1. Pyrophosphate and BP structures.
Article
Cite This: ACS Omega 2018, 3, 14581469
© 2018 American Chemical Society 1458 DOI: 10.1021/acsomega.7b01686
ACS Omega 2018, 3, 14581469
This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.
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. Poly-
MA-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.
2. RESULTS AND DISCUSSION
2.1. 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 2. 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 γposition can be achieved while
protecting the carboxylic acid in the αposition. 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
2.2. 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 3A).
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 3B),
and a hydrodynamic diameter of 163.2 ±7nm(Figure 3C).
The reason for the dry diameter of the NPs being signicantly
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
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 4 exhibits the ζ-potential curve of the
dispersed polyMA-Glu-BP NPs (0.1 mg/mL) at dierent pH
values. At pH 2.5, the graph exhibits a low positive ζ-potential,
which may be explained by the presence of protonated amine
Figure 2. Synthesis of the MA-Glu-BP vinylic monomer.
Figure 3. Preparation scheme (A), TEM image (B), and hydro-
dynamic size histogram (C) of polyMA-Glu-BP NPs.
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groups on the surface of the NPs. From pH 2.5 to 10.9, the
curve shows a signicant 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
aζ-potential of 24.3 ±0.9 mV. The isoelectric point
calculated was 2.9.
2.3. 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 5A 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.
3840
To determine the optimal reaction time for the polymer-
ization of the polyMA-Glu-BP NPs, the samples were puried,
lyophilized, and weighed. The yield at various time points was
calculated (Figure 5B). 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.
2.4. Eect of the Polymerization Parameters on the
Size and Size Distribution of the Formed PolyMA-Glu-
BP NPs. 2.4.1. Eect of the Total Monomer Concentration.
Figure 6 illustrates the eect 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
dierent total concentrations, while the other reaction
conditions remain untouched.
It is evident, as shown in Figure 6, 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
ndings can be attributed to the increasing total amount of
polymer formed in the reaction as well as the accelerating
Figure 4. ζpotential of polyMA-Glu-BP NPs at various pH values.
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.
Figure 6. Relationship between the total monomer concentration and
the diameter size and size distribution of the formed particles.
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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 agglomer-
ation of the formed particles and therefore an increase in the
particle diameter. A similar behavior on the eect of the total
monomer concentration on the size and size distribution of
various NPs was already reported in the literature.
4143
2.4.2. Eect of the Cross-Linker Concentration. Figure 7
displays the inuence 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).
Each sample 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 7 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 diculties 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
dicult with higher particle cross-linkage, as already indicated
in the literature.
4446
2.4.3. Eect of Mwand Concentration of the Stabilizer
Polyvinylpyrrolidone. Figure 8 illustrates the eect 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
dierence 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.
4750
The same reason may also explain
the signicantly 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 8.
2.4.4. Eect of the Initiator Concentration. Figure 9
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
Figure 7. Eect of the weight % ratio [TTEGDA]/([MA-Glu-BP] +
[APMA]) on the diameter and size distribution of the formed
polyMA-Glu-BP NPs.
Figure 8. Eect 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.
Figure 9. Eect of the concentration of PPS on the diameter of
formed particles.
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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 polymer-
ization sites; hence more particles of smaller diameter are
formed, as reported in the literarure.
44,48,51
2.4.5. Eect of Temperature. Figure 10 exhibits the eect of
the reaction temperature on the hydrodynamic diameter and
size distribution of the formed particles. The gure
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 ndings could be explained by the fact that the
increase in temperature causes an increase in the decom-
position 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
2.5. Calcium Anity Test Using Coral. Coral skeletons
contain large amounts of calcium carbonate (CaCO3),
52,53
making their fragments a useful tool for in vitro calcium anity
testing. Therefore, the adsorption of the near-infrared (NIR)
uorescent polyMA-Glu-BP NPs compared with NIR uo-
rescent control NPs (containing methacryloylglutamine mono-
mer instead of the MA-Glu-BP monomer) toward coral was
demonstrated using a NIR uorescence microscope (Figure
11). Corals treated with 0.5 mg/mL polyMA-Glu-BP NPs
(Figure 11F) exhibit a prominent uorescence compared to
corals treated with 0.5 mg/mL control NPs (Figure 11D),
whichdonotexhibitanynoticeableuorescence. The
uorescence 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 uorescence
intensity than the control NPs.
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 12. There is a distinct
dierence between the surfaces of the various treated corals.
The surface of the corals treated with water (Figure 12A) and
control NPs (Figure 12B) appear similar and are composed of
spikey panels. In contrast, the corals treated with the polyMA-
Glu-BP NPs (Figure 12C) exhibit a smooth surface, indicating
that the surface of the coral is coated by the NPs. These results
can be attributed to the anity of the BP groups to the calcium
ions in the coral in comparison to the control NPs. These
ndings conrm that the polyMA-Glu-BP NPs have strong
anity toward calcium in comparison to the control NPs.
2.6. Toxicity of the PolyMA-Glu-BP NPs. Cytotoxicity
and toxicity assays [lactate dehydrogenase (LDH) and 2,3-bis-
(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxani-
lide (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 13. The
cells were treated for 48 and 72 h with a serial diluted
concentration of the polyMA-Glu-BP NP aqueous dispersion
(11000 μg/mL). The LDH results obtained (Figure 13A)
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 13B). The XTT
results in Figure 13C,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.
2.7. Cellular Uptake of Cy7-polyMA-Glu-BP NPs by
J774A1 Macrophages. To perform kinetic cellular uptake
studies of the polyMA-Glu-BP NPs, the uorescent 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 14). The results summarized in Table 1 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 uorescence (99.55 ±0.07%).
The uorescence values remain similar for the following hours:
99.9 ±0% after 5 h and 99.95 ±0.07% after 24 h.
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 2). Notably,
neither of the tested concentrations demonstrated total kill of S.
aureus. Nevertheless, at the range 7.50.23 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.
3. 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 inuence of the following parameters on
the diameter of the polyMA-Glu-BP NPs was studied: total
monomer concentration, cross-linker concentration, stabilizer
Mwand concentration, initiator concentration, reaction temper-
ature, 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.
Figure 10. Eect of the reaction temperature on the diameter of the
formed particles.
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The surface BP functional groups have a high anity 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 anity 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 eect 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 ndings 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 signicantly 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
dierent 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 eect toward
bacteria.
Figure 11. Fluorescence microscopy images: bright-eld images of the corals treated with water (A), 0.5 mg/mL NIR uorescent control NPs (C),
and 0.5 mg/mL NIR uorescent polyMA-Glu-BP NPs (E) and NIR images of the corals treated with water (B), 0.5 mg/mL NIR uorescent control
NPs (D), and 0.5 mg/mL NIR uorescent polyMA-Glu-BP NPs (F).
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.
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4. EXPERIMENTAL SECTION
4.1. Materials. The following analytical-grade chemicals
were purchased from commercial sources and used without
further purication: glutamic acid, acetic anhydride, phthalic
anhydride, TTEGDA, PPS, PVP (Mw40k and Mw360k),
sodium hydroxide (1 N), hydrochloric acid (1 N), anhydrous
dichloromethane, anhydrous tetrahydrofuran (THF), anhy-
drous methanol, anhydrous dimethyl sulfoxide (DMSO), O-
[(N-succinimidyl)succinyl-aminoethyl-O-methylpolyethylene
glycol (PEG-NHS, Mw750), methacryloyl chloride, tris-
(trimethylsilyl)phosphite, and glutamine from Sigma (Rehovot,
Israel); APMA from Polysciences, Inc. (Warrington, PA);
dialysis membrane (1000k16 mm), sodium carbonate, and
sodium bicarbonate from Bio-Lab Ltd. (Jerusalem, Israel);
cyanine7 N-hydroxysuccinimide (Cy7-NHS) ester from Lumip-
robe Corporation (Florida, USA); Dulbeccos phosphate-
buered saline (PBS), Dulbeccos modied Eagles 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 puried by passing deionized DDW
through an Elgastat Spectrum reverse osmosis system (ELGA
Ltd., High Wycombe, UK). Coral scaold was received as a gift
from Boneus Ltd. (Haifa, Israel).
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.
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 Times
a
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
a
Antibacterial activity of the polyMA-Glu-BP NPs.
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
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4.2. Synthesis of the Vinylic Monomer MA-Glu-BP. 1H
NMR, 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 infrared (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 cm1resolution.
Elemental analysis was performed with a PerkinElmer 2400
series II analyzer, by the analytical laboratories of the Hebrew
University, Jerusalem.
Low-resolution mass spectra were obtained on a Micromass
Q-Tof microspectrometer in the electrospray mode.
4.2.1. Synthesis and Characterization of N-Phthaloyl
Glutamic Acid. N-Phthaloyl 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 ask
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
ltered and dried to yield the desired product (10.7 g, 44.4%
yield).
1H NMR (DMSO-d6) 300 MHz: 2.212.39 (m, 4H,
HO2CCH2CH2), 4.784.83 (m, 1H, NCHCO2H),
7.857.92 (m, 4H, ArH).
TOF MS+(m/z): [M + Na]+calcd for C13H11NO6Na+,
300.05; found, 300.
4.2.2. Synthesis and Characterization of N-Phthaloyl
Glutamic Anhydride. N-Phthaloyl glutamic anhydride was
synthesized similar 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).
1H NMR (DMSO-d6) 300 MHz: 2.122.18 (m, 1H, N
CHCH2CH2CO2), 2.562.71 (m, 1H, NCHCH2
CH2CO2), 2.963.02 (m, 1H, NCHCH2CH2
CO2), 3.083.20 (m, 1H, NCHCH2CH2CO2),
5.455.52 (m, 1H, NCHCO2), 7.877.95 (m, 4H, Ar
H).
TOF MS+(m/z): [M + H]+calcd for C13H10NO5+, 260.06;
found, 260.
4.2.3. Synthesis and Characterization of N-Pht-Glu-BP. N-
Phthaloyl glutamic anhydride (8 g, 0.031 mol) was dissolved in
200 mL of dry THF under a nitrogen atmosphere to give a
clear o-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 evapo-
rated, 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
1H NMR (D2O) 600 MHz: 1.862.05 (m, 2H, PCCH2),
2.362.52 (m, 2H, PCCH2CH2) 4.854.89 (m, 1H, N
CHCO2H), 7.757.83 (m, 4H, ArH). 13C NMR (D2O)
600 MHz: 24.26 (t, 3Jcp = 7 Hz, PCCH2CH2), 31.08 (s,
PCCH2), 53.48 (s, NCHCO2H), 73.89 (t, 1Jcp = 138
Hz, PCP), 124.32 (s, COAr ortho), 131.66 (s, COAr
ipso), 135.58 (s, COAr meta), 170.43 (s, ArCON), 173.99
(s, CO2H). 31P NMR (D2O) 400 MHz: 19.13 (s).
TOF MS(m/z): [M H]calcd for C13H14NO11P2
,
422.2; found, 422.
FTIR (KBr): 3429 (COOH), 1773 (COOH), 1711 (Ar
CO), 1396 (CO2
), 1174 (PO), 1070 (PCOH), 930
(COOH dimer), 722 (PC), 532 (OPO).
Elemental analysis: Elemental analysis calcd 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.
Decomposes at 290 °C.
4.2.4. Synthesis and Characterization of γ-Glu-BP. N-Pht-
Glu-BP (10.8 g, 0.036 mol) was dissolved in 6 M HCl solution
(180 mL), and the clear colorless solution was reuxed
overnight. The solution was cooled to rt and then refrigerated
for 4 h. The resulting white crystals of phthalic acid were
ltered, and the clear colorless ltrate was evaporated to yield
the desired compound as a white solid (5.9 g, 98.7% yield).
34
1H NMR (D2O) 400 MHz: 2.032.34 (m, 4H, PCCH2
CH2), 4.064.09 (m, 1H, NCHCO2H). 13C NMR (D2O)
400 MHz: 25.66 (t, 3Jcp = 9 Hz, PCCH2CH2), 29.76 (s,
PCCH2), 54.01 (s, NH2CHCO2H), 73.53 (t, 1Jcp = 183
Hz, PCP), 173.04 (s, CO2H). 31P NMR (D2O) 400 MHz:
18.74 (s).
TOF MS+(m/z): [M + H]+calcd for C5H14NO9P2+, 294.01;
found, 294.
FTIR (KBr): 3170 (COOH), 1732 (COOH), 1632 (HN),
1528 (CO2
), 1162 (PO), 1064 (PCOH), 919 (COOH
dimer), 529 (OPO).
Elemental analysis: elemental analysis calcd 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.
Decomposes at 290 °C.
4.2.5. Synthesis and Characterization of the Monomer
MA-Glu-BP. Methacryloyl chloride (1 mL, 0.01 mol) was added
dropwise to a solution of γ-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 ltration and
washed with additional ethanol (850 mL), yielding the desired
product as a salt (2.5 g, 76.0% yield).
35,36
1H NMR (D2O) 400 MHz: 1.842.21 (m, 4H, PCCH2
CH2), 1.96 (s, 3H, CH2CCH3), 4.094.12 (m, 1H, N
CHCO2H), 5.47 (s, 1H, cis CH2CCH3), 5.77 (s, 1H,
trans CH2CH3). 13C NMR (D2O) 400 MHz: 18.48 (s,
CH2CCH3), 27.3927.52 (t, 3JCP = 24 Hz, PCCH2
CH2), 32.75 (s, PCCH2), 57.53 (s, NHCHCO2H),
74.2476.83 (t, 1JCP = 130 Hz, PCP), 121.58 (s, CH2C
CH3), 139.87 (s, CH2CCH3), 172.44 (s, CONH), 180.53
(s, CO2H). 31P NMR (D2O) 400 MHz: 18.7819.38 (q, JPP =
19.96 Hz).
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TOF MS(m/z): [M H]calcd for C9H16NO10P2
,
360.03; found, 360.
FTIR (KBr): 3406 (HN), 2233 (CC), 1655 (CON
H), 1594 (CO2), 1455 (CH2), 1408 (CH3), 1222 (PO),
1107 (PCOH), 1003 (COOH dimer), 957 (CCH2), 557
(OPO).
Elemental analysis: elemental analysis calcd 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.
Decomposes at 290 °C.
4.3. Synthesis of the Cross-Linked PolyMA-Glu-BP
NPs. MA-Glu-BP (17.5 mg), 5 mg of 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 N2to 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-oof 1 000 000k) with DDW,
giving particles with a hydrodynamic diameter of 163.2 ±7nm
and a dry diameter of 61.2 ±6 nm.
The NP formation yield, 33.12%, was calculated by the
following expression:
WWNanoparticles yield ( )/( ) 100
particles total monomers
where Wparticles is the weight of the dried particles and
Wtotal monomers is the initial weight of the three polymerized
monomers.
The concentration of the BP groups of the particles, as
calculated from the elemental analysis, was 0.2 mmol/g
particles.
Elemental analysis: C, 52.84% (calcd 50.32%); H, 7.02%
(calcd 6.95%); N, 1.31% (calcd 4.08%); P, 0.59% (calcd 4%).
4.4. 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).
Electrokinetic 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 analytical laboratories of the Hebrew
University, Jerusalem. Phosphorus content was determined
using the oxygen ask combustion method followed by ion
chromatography analysis using a Dionex IC system.
4.5. 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. Briey, 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 uorescent polyMA-Glu-BP NP aqueous
dispersion and stirred at rt for an additional 1 h. The obtained
NIR uorescent conjugated polyMA-Glu-BP NPs were then
washed of excess reagents by extensive dialysis in DDW (a
cutoof 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.
4.6. 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
ltration. The aqueous ltrate was acidied to pH 1 with HCl
32% until a white precipitant appeared. The precipitant was
ltered o, 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
1H NMR (D2O) 300 MHz: 1.99 (s, 3H, CH2CCH3),
2.052.34 (m, 2H, H2NCOCH2CH2), 2.432.48 (t,
2H, H2NCOCH2CH2), 4.434.48 (quart, 1H, NCH
CO2H), 5.56 (s, 1H, cis CH2CCH3), 5.79 (s, 1H, trans
CH2CH3).
13C NMR (D2O) 400 MHz: 18.40 (s, CH2CCH3), 27.13
(s, H2NCOCH2CH2), 32.75 (s, H2NCOCH2
CH2), 53.68 (s, NHCHCO2H), 122.43 (s, CH2C
CH3), 139.45 (s, CH2CCH3), 172.65 (s, CONH), 176.39
(s, H2NCOCH2CH2), 178.92 (s, CO2H).
4.7. Synthesis of the Control NPs. Methacryloylglut-
amine (70 mg), 20 mg of 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 cutoof 1 000 000k) with DDW, giving
particles with a hydrodynamic diameter of 149.1 ±36 nm.
The NP formation yield, 65.92%, was calculated by the
following expression:
WWNanoparticles yield ( )/( ) 100
particles total monomers
where Wparticles is the weight of the dried particles and
Wtotal monomers is the initial weight of the three polymerized
monomers.
4.8. 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. Briey, 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 (Mw750) to the NIR uorescent control NP
aqueous dispersion and stirred at rt for an additional 1 h. The
obtained NIR uorescent conjugated control NPs were then
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washed of excess reagents by extensive dialysis in DDW (a
cutoof 1 000 000k).
4.9. Calcium Anity Test of the PolyMA-Glu-BP NPs.
The anity of the polyMA-Glu-BP NPs to calcium ions was
evaluated by incubating coral fragments (5.55 mg) with NIR
uorescent 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 uorescent
control NP dispersion (200 μL, 0.5 mg/mL) and DDW. The
coral fragments were evaluated by a uorescent 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).
4.10. 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 Dulbeccos modied Eagles 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 Hams F12
medium and Dulbeccos modied Eagles 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
manufacturers instructions.
55
Briey, cells (5 ×103cells 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 polyMA-Glu-BP NPs were freshly dispersed in PBS
(1.25 and 2.5 mg/mL) and then added to the 95% conuent
cell culture in culture medium. The cell cultures were further
incubated at 37 °C in a humidied 5% CO2incubator and then
checked for cellular cytotoxicity after 48 h of incubation. The
percentage of cell cytotoxicity was calculated using the formula
shown in the manufacturers protocol.
55
All samples were tested
in tetraplicates.
4.11. Cell Proliferation Analysis. XTT analysis
56
was
performed according to the manufacturers instructions (Bio-
logical 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 °Cin5%CO
2.
The polyMA-Glu-BP NPs were freshly dispersed in PBS (1.25
and 2.5 mg/mL) and then added to the 95% conuent cell
culture in culture medium. The cell cultures were further
incubated at 37 °C in a humidied 5% CO2incubator and then
checked for cellular viability after 48 h of incubation. The
percentage of cell viability was calculated using the formula
shown in the manufacturers protocol.
56
All samples were tested
in tetraplicates.
4.12. Flow Cytometry Analyses of NP Uptake. To study
the eect 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 ow 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
lter. Data were processed by FlowJo v7.6.4.
57
4.13. Bacterial Cultures and Growth Conditions.
Bacterial 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.
4.14. 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 105colony-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.
AUTHOR INFORMATION
Corresponding Author
*E-mail: shlomo.margel@mail.biu.ac.il (S.M.).
ORCID
Shlomo Margel: 0000-0001-6524-8179
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the nal version of
the manuscript.
Funding
This research did not receive any specic grant from funding
agencies in the public, commercial, or not-for-prot sectors.
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Norma Marcus-Rudnick
for her assistance in editing. The authors would like to thank
Elad Hadad for his assistance with the HR-SEM images. The
authors would like to thank Hai Haham for his assistance with
the TEM images. The authors would like to thank Gila Jacobi
for helping with the biological experiments.
ACS Omega Article
DOI: 10.1021/acsomega.7b01686
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1467
ABBREVIATIONS
BPs, bisphosphonates; NPs, nanoparticles; N-Pht-Glu-BP, N-
phthaloyl glutamate bisphosphonate; γ-Glu-BP, gamma gluta-
mate bisphosphonate; MA-Glu-BP, methacrylate glutamate
bisphosphonate; polyMA-Glu-BP NPs, polymeric methacrylate
glutamate bisphosphonate nanoparticles; APMA, N-(3-amino-
propyl) methacrylamide hydrochloride; Cy7, cyanine7; Cy7-
NHS, cyanine7 N-hydroxysuccinimide; TTEGDA, tetra ethyl-
ene glycol diacrylate; PVP, polyvinylpyrrolidone; PPS,
potassium persulfate; PEG-NHS, O-[(N-succinimidyl)-
succinyl-aminoethyl-O-methylpolyethylene glycol; PBS, phos-
phate-buered saline; DMEM, Dulbeccos modied Eagles
medium; FBS, fetal bovine serum; DDW, double distilled
water; HCl, hydrochloric acid; THF, tetrahydrofuran; TEM,
transmission electron microscopy; DMSO, dimethyl sulfoxide;
BB, sodium bicarbonate; NIR, near-infrared; HR-SEM, high-
resolution scanning electron microscope; LDH, lactate
dehydrogenase; XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophen-
yl)-2H-tetrazolium-5-carboxanilide; CO2, carbon dioxide;
MBC, minimum bactericidal concentration; CaCO3, calcium
carbonate; Cy7-polyMA-Glu-BP NPs, cyanine7-conjugated
polymeric methacrylate glutamate bisphosphonate nanopar-
ticles
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... Here we present the synthesis of aqueous dispersed poly(StBP) NPs with a high surface area, which have a significant antibacterial activity, and may be useful as antifouling coating with a better uniform coverage than the grafted bisphosphonate chains. In addition, the synthesis of these cross-linked poly(StBP) NPs produced a higher yield (86%) than other bisphosphonate NPs previously published [26] and contain an aromatic ring which enhances the NPs rigidity. Furthermore, these poly(StBP) NPs may be useful in various applications such as dental devices and bone tissue engineering. ...
... For example, in the presence of 0.5, 1.5, and 2.5% (w/ v) TEGDMA ([TEGDMA]/( [StBP] + [APMA]) = 0.1, 0.4, and 1.0% (w/ w), respectively), the diameter and size distribution of the cross-linked poly(StBP) NPs decreased from 286 ± 28 to 222 ± 28 and to 149 ± 16 nm, respectively. The reason may be that as the cross-linking increases, the propensity to monomer swelling of the growing nuclei decreases, resulting in smaller particles [24,26]. By increasing the presence of the cross-linking monomer TEGDMA above 2.5% (w/v), smaller NPs are produced, however, the concentration of the functional groups (bisphosphonate and primary amino groups) in the NPs decreases. ...
... These results could be explained by the fast formation of a cross-linked polymer network in the early stages of the reaction, which forms the initial particles. As the polymerization progresses, the cross-linkage increases, and so the NPs become denser and decrease in diameter [26,38]. ...
... From a clinical point of view, the studies concerning the use of BPs in the treatment of some protozoal infections [48], in the modulation of the immune response [49], in the mechanisms of tissue repair [50], and generally in improving cell survival [51], could have a significant impact on the management of many diseases. Additionally, in the field of bone health, new research addressed the role of BPs as transporters of other pharmacological substances that would otherwise reach the bone tissue with difficulty and low doses [52][53][54][55]. ...
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Book
There is an enormous quantity of literature, including numerous patents, on the functions and applications of phosphates in foods. No attempt has been made to review every publication, as such an exhaustive review would require a sizeable book. Anyone interested in further details on any topic covered in this treatise should consult the references listed at the end of the chapter. The bibliographies provided in the references for each topic should provide more thorough coverage. It is hoped that the references cited are those with the most useful information on the phosphate applications and their effects on foods.
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There has been increased concern during the past few decades over the role bacterial biofilms play in causing a variety of health problems, especially since they exhibit a high degree of resistance to antibiotics and are able to survive in hostile environments. Biofilms consist of bacterial aggregates enveloped by a self-produced matrix attached to the surface. Ca(2+) ions promote the formation of biofilms, and enhance their stability, viscosity, and strength. Bisphosphonates exhibit a high affinity for Ca(2+) ions, and may inhibit the formation of biofilms by acting as sequestering agents for Ca(2+) ions. Although the antibacterial activity of bisphosphonates is well known, research into their anti-biofilm behavior is still in its early stages. In this study, we describe the synthesis of a new thin coating composed of poly(styryl bisphosphonate) grafted onto oxidized polypropylene films for anti-biofilm applications. This grafting process was performed by graft polymerization of styryl bisphosphonate vinylic monomer onto O2 plasma-treated polypropylene films. The surface modification of the polypropylene films was confirmed using surface measurements, including X-ray photoelectron spectroscopy, atomic force microscopy, and water contact angle goniometry. Significant inhibition of biofilm formation was achieved for both Gram-negative and Gram-positive bacteria.
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The vinylic monomer methylstyrene farmin (MSF) was synthesized by interacting p-chloromethyl styrene with farmin in basic distilled water. Crosslinked poly(methyl styrene farmin) nanoparticles (PMSF NPs) were then synthesized by dispersion co-polymerization of the MSF monomer with the crosslinking monomer tetra(ethylene glycol) diacrylate (TTEGDA) in an aqueous continuous phase. The effect of various polymerization parameters on the size and size distribution of the formed NPs has been elucidated. These PMSF NPs contain quaternary ammonium groups and long alkyl chain groups, and therefore are expected to possess efficient antibacterial and antibiofilm properties. The antibacterial and antibiofilm properties of PMSF NPs of 40 ± 9 nm and NPs-coated films were demonstrated. Due to their promising antibacterial and antibiofilm properties, these newly NPs could be utilized in a variety of industrial applications as a new type of biocide and antibiofilm additive.
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A facile controlled-release nanogels delivery system has been developed by using hydroxypropyl methylcellulose (HPMC) hybrid nanogels as encapsulation shell materials, which were synthesized by surfactant-free polymerization in aqueous solution. The effects of reaction time and cross-linker concentration on the size of the nanogels have been studied. The results showed that in a certain range, the particle size decreased with increasing reaction time and increasing concentration of cross-linker. Meanwhile, at the feeding ratio 0.05/1 of HPMC/methacrylic acid (MAA), the LCST of prepared nanogels at pH = 6 was close to the body temperature, which can be used as sustained insulin delivery system. Besides, the HPMC nanogels loaded with insulin had a high drug loading of 21.3% and a high entrapment efficiency of 95.7%. The release behavior of the insulin nanogels can be adjusted by pH and temperature which will have potential applications in controlled release delivery system.
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Although much progress has been made in the field of cancer therapy, cancer remains one of the leading causes of death in the western world. Here we have designed and studied a unique type of composite multi-functional near IR (NIR) fluorescent iron oxide (IO) nanoparticles (NPs) of narrow size distribution for tumor targeting and therapy. These NPs were prepared by nucleation followed by controlled growth of thin films of IO onto Cy7-conjugated gelatin nuclei and coated with human serum albumin (HSA) by a thermal precipitation process. The hydrodynamic diameter of these core-shell NPs could be easily controlled by altering the precipitation reaction temperature. For targeting and an anti-cancer effect, we conjugated the Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) cytokine to the surface of the NIR fluorescent IO/HSA NPs via a polyethylene glycol (3 kDa) linker. The conjugated TRAIL exhibited enhanced and prolonged anti-cancer activity in both human glioblastoma multiforme and colon cancer cell lines. Further, the combination of these IO/HSA-TRAIL NPs with the commonly used chemotherapeutic drug doxorubicin resulted in a synergistic anti-cancer effect on these cancer cell lines. In addition, we also clearly demonstrated by topically and IV administrations the specific targeting effect and the synergistic therapy effect of the NIR fluorescent NPs in-ovo, by using a chicken embryo model of tumors derived from the various human cancer cell lines.