Synthesis and bactericidal evaluation of imide N-halamine-loaded PMMA nanoparticles

Article abstract of DOI:10.1039/c4nj01806k

Imide N-halamine-loaded poly(methyl methacrylate) nanoparticles (PMMA) based on barbituric acid were synthesized as novel antimicrobial agents using radical copolymerization. Evidence for loading imide N-halamine on PMMA nanoparticles has been inferred from different techniques like ¹H NMR, FTIR, TEM, SEM, and XPS analyses. The sterilizing effect of the products on bacterial strains was systematically evaluated by selecting Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa as model pathogenic bacteria. The zone of inhibition study and the spread plate technique suggested that the imide N-halamine-loaded PMMA nanoparticles possessed powerful bactericidal activity towards both Gram-positive and Gram-negative bacteria. The effects of contact period, N-halamine structure, particle size, and chlorine content on biocidal efficiency were investigated as well. Long-term stability of the imide N-halamine-loaded PMMA nanoparticles was also confirmed as a function of storage period.

Full text of DOI:10.1039/c4nj01806k

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Synthesis and bactericidal evaluation of imide
N-halamine-loaded PMMA nanoparticles
Cite this:DOI: 10.1039/c4nj01806k
Qigeqi Dong,†ab Qian Cai,†a Yangyang Gao,a Shiqi Zhang,c Ge Gao,d
Chokto Harnoode,a Morigenb and Alideertu Dong*a
Imide N-halamine-loaded poly(methyl methacrylate) nanoparticles (PMMA) based on barbituric acid were
synthesized as novel antimicrobial agents using radical copolymerization. Evidence for loading imide
N-halamine on PMMA nanoparticles has been inferred from different techniques like 1H NMR, FTIR, TEM,
SEM, and XPS analyses. The sterilizing effect of the products on bacterial strains was systematically
evaluated by selecting Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa as model
pathogenic bacteria. The zone of inhibition study and the spread plate technique suggested that the imide
N-halamine-loaded PMMA nanoparticles possessed powerful bactericidal activity towards both Gram-
positive and Gram-negative bacteria. The effects of contact period, N-halamine structure, particle size,
and chlorine content on biocidal efficiency were investigated as well. Long-term stability of the imide
N-halamine-loaded PMMA nanoparticles was also confirmed as a function of storage period.
Received (in Montpellier, France)
15th October 2014,
Accepted 12th December 2014
DOI: 10.1039/c4nj01806k
www.rsc.org/njc
N-halamine polymers from hydantoin-based monomers with
1. Introduction
outstanding antibacterial capability.14–16 Liang’s group loaded qua-
Environmental contamination induced by pathogenic microor- ternarized N-halamine on cellulose and demonstrated excellent
ganisms is of great concern in the medical field, public hygiene, antimicrobial activity, washing durability, and storage stability.17–19
food safety control, and water purification, etc.1,2 Various kinds of Numerous studies have shown that the bactericidal activity
bactericides thus have been developed in recent decades.3–6 of N-halamine is in the order of imide 4 amide 4 amine
Among the various bactericides, considerable research efforts N-halamine.20 Therefore, developing N-halamines with more
have been devoted to fabricating N-halamines due to their instant imide structure to enhance antimicrobial efficiency is advisable.
and total sterilization of a wide range of microorganisms.7 Barbituric acid, which is well known for its medical and biolo-
N-Halamines possess several major advantages such as long- gical functions, is a promising heterocyclic compounds with two
term storage, high stability, and regenerability.8 Moreover, unlike imide functional groups in the structure.21 Imide group can
inorganic halogens, N-halamines are durable, less corrosive, and readily transfer to N–Cl structure upon chlorination, by which
do not decompose in water to form toxic products.9
imide type N-halamines as a potential biocide can be achieved.
N-Halamines with one or more nitrogen–halogen covalent bond Nevertheless, reports on N-halamines based on barbituric acid
are generally synthesized through the halogenation of the corres- are quite rare.22,23
ponding amide, imide, or amino group.10 5,5-Dimethylhydantoin
The antibacterial performance of N-halamines is strongly
with one amide and one imide commonly accepted as N-halamine dependent on their surface area, and thus reducing materials’
precursor has been widely reported. Worley et al. designed size to improve surface area is a promising idea.24 Numerous
numerous N-halamines from 5,5-dimethylhydantoin with power- reports have confirmed that nanoparticles exhibit high anti-
ful antimicrobial activity against both Gram-positive and Gram- bacterial activity compared with their bulk counterparts owing
negative bacteria.11–13 Sun et al. developed various kinds of to their larger activated surface area providing more active sites
for deactivating pathogenic bacteria.25 Selecting chemically
inert nanoparticles with controllable size as solid supports to
enhance the activated surface is most widely utilized. In our
previous studies, several N-halamine antibacterial nano-
particles were prepared with silica, iron oxide, polystyrene,
and poly(styrene-co-acrylic acid) nanoparticles as templates,
respectively.26–32 The introduction of these templates can
achieve the aim of improving the activated surface, but there
are also the unavoidable drawbacks of the cumbersome
a College of Chemistry and Chemical Engineering, Inner Mongolia University,
Hohhot 010021, People’s Republic of China. E-mail: dongali@imu.edu.cn
b College of Life Science, Inner Mongolia University, Hohhot 010021,
People’s Republic of China
c PhD School of Materiaux, Mechanics, Environnement, Energy, Process and
Production Engineering (I-MEP2), University of Grenoble, Grenoble 38031, France
d College of Chemistry, Jilin University, Changchun 130021,
People’s Republic of China
† Contributed equally to this work.
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synthesis procedures required by more reacting steps and the (hr = 1253.6 eV). The X-ray anode was run at 250 W, and the
pollution caused by the templates during the practical applica- high voltage was kept at 14.0 kV with a detection angle at 540.
tion. Designing N-halamine nanoparticles without using solid All the binding energies were calibrated by using the contain-
templates was subsequently investigated by our group, and the ment carbon (C 1s = 284.6 eV). SEM images were taken on a
study for synthesizing N-halamine homopolymer nanoparticles Shimadzu SSX-550 field emission scanning electron micro-
via the radical polymerization of allyl monomer was thus scope at 15.0 kV.
carried out. Unfortunately, N-halamine homopolymers were
2.3 Preparation of 5-allylbarbituric acid
hardly obtained because of the radical autoinhibition effect of
the allylic structure.33 In response to such difficulties, methyl
The suspension of 0.92 g sodium hydride in 50 mL tetrahydro-
methacrylate favorable to radical polymerization was chosen to furan was added dropwise into 3.20 g diethyl malonate at 0 1C.
copolymerize with N-halamine monomer to obtain antibacterial
polymer with higher molecular weight.34 Few published studies
have concerned the preparation of functionalized PMMA nano-
particles through a one-step process by using the radical copoly-
merization of MMA with a functional component.35,36
The reaction mixture was warmed to room temperature and
stirred for 1 h. 2.90 g of allyl bromide was added, and the
resulting solution was stirred overnight. After the addition of
100 mL ultrapure water and extraction with diethyl ether, the
organic layer was washed with brine, dried with magnesium
sulfate and concentrated under reduced pressure. The crude pro-
duct was purified by column chromatography (hexane/diethyl ether,
We report herein the facile synthesis of novel imide
N-halamine nanoparticles as a promising antibacterial agent
through the functionalization of PMMA nanoparticles with 10 : 1) to afford 3.44 g diethyl allylmalonate. A mixture of dry sodium
barbituric acid-based N-halamine without using additional
template nanoparticles. Imide N-halamine-loaded PMMA nano-
particles were characterized with different techniques such as
transmission electron microscopy (TEM), scanning electron
microscopy (SEM), nuclear magnetic resonance (NMR), Fourier
transform infrared (FTIR), and X-ray photoelectron spectroscopy
(XPS). To evaluate the bactericidal capability, Staphylococcus
aureus (Gram-positive), Escherichia coli (Gram-negative), and
Pseudomonas aeruginosa (Gram-negative) were selected as model
bacteria. The antimicrobial results demonstrated that the
as-synthesized imide N-halamine-loaded PMMA nanoparticles
possessed excellent biocidal activity against both Gram-positive
and Gram-negative bacteria.
methoxide (prepared from 0.2875 g sodium and 7.5 mL methanol),
0.75 g urea, 2.5 g diethyl allylmalonate, and 2.5 mL acetone was
stirred under reflux for 7 h. The precipitate was collected by
filtration, washed with acetone, suspended in 5 mL water, and
acidified with concentrated aqueous hydrochloric acid to pH 1–2.
The precipitate was filtered off and recrystallized from ethanol to
obtain 5-allylbarbituric acid (ABBA) as colorless needles.37,38
2.4 Preparation of poly(ABBA-co-MMA) nanoparticles
Sodium bicarbonate, potassium persulfate, and 100 mL ultra-
pure water were added in a 250 mL three-necked flask with a
condenser and an N2 gas inlet, and a mixture of ABBA dissolved
in methyl methacrylate was added. The reaction mixture was
maintained at 75 1C with stirring for 24 h. Copolymer nano-
particles with different particle sizes (523.81 nm, 809.52 nm,
904.76 nm, and 976.19 nm) were fabricated at different mono-
mer concentrations (1.0 wt%, 2.0 wt%, 3.0 wt%, and 5.0 wt%).
2. Experimental section
2.1 Materials
Sodium and urea were obtained from Nanjing Chemical
Reagent Co., Ltd. Anhydrous methanol, hydrochloric acid,
hexane, tetrahydrofuran, diethyl ether, and acetone were pur-
chased from Beijing Chemical Company. Methyl methacrylate
and potassium persulfate were obtained from Tianjin Chemical
Reagent Plant and Shanghai Chemical Reagent Plant, respec-
tively. Sodium hydride was obtained from Beijing Hengye
Zhongyuan Chemical Co., Ltd. Diethyl malonate, allyl bromide,
magnesium sulfate, sodium chloride, sodium bicarbonate, and
sodium hypochlorite were obtained from Sinopharm Chemical
Reagent Co., Ltd. The other reagents were analytical grade and
were used without any purification.
2.5 Preparation of imide N-halamine-loaded PMMA
nanoparticles
Poly(ABBA-co-MMA) nanoparticles were immersed in a 10%
commercial aqueous sodium hypochlorite solution buffered
at pH 7 at room temperature for 12 h. The imide N-halamine-
loaded PMMA nanoparticles were washed thoroughly with
ultrapure water and dried at 40 1C for 6 h to remove any
remaining free chlorine from the surface of the sample.16–18
2.6 Determination of chlorine content
The active chlorine content of the imide N-halamine-loaded
PMMA nanoparticles was determined by the iodometric/
thiosulfate titration procedure.9–16 The weight percentage of
chlorine (Cl%) for the sample was calculated according to the
following equation:9,16
2.2 Characterization
1H NMR spectra were recorded on a Bruker AV300 instrument.
FTIR spectra were captured by using a Thermo Nicolet
(Woburn, MA) Avatar 370 FTIR spectrometer. TEM images were
taken on a Hitachi H-8100 transmission electron microscope at
35:5 ðVCl ꢁ V0Þ ꢀ 10ꢁ3 ꢀ 0:01
Clð%Þ ¼
ꢀ
ꢀ 100
2
WCl
200 kV. X-ray photoelectron spectra (XPS) measurements were where VCl and V0 are the volumes (mL) of sodium thiosulfate
carried out on a PHI-5000CESCA system with Mg K radiation solutions consumed in the titration of the chlorinated and
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unchlorinated samples, respectively, and WCl is the mass of the
chlorinated sample (g).
2.7 Antibacterial test
The antibacterial behavior of the product was assessed by
a modified Kirby–Bauer (KB) technique.39 The surface of
Luria-Bertani agar plate and tryptic soy agar plate was overlaid
with 1 mL of 108–9 CFU per mL of E. coli (ATCC 8099, Gram-
negative bacteria). The plates were then allowed to stand at
37 1C for 4 h. The imide N-halamine-loaded PMMA nano-
particles were placed onto the surface of each of the bacteria-
containing agar plates, and gently pressed with a sterile forceps
to ensure full contact between the sample and the agar. The
same procedure was also applied to the pure PMMA nano-
particles as control. After incubation at 37 1C for 24 h, the
inhibition zone around the sample was measured.
S. aureus (ATCC 25923, Gram-positive bacteria) and
P. aeruginosa (ATCC 27853, Gram-negative bacteria) were used
as model microorganisms to test the antibacterial activities of
the samples. Bacteria were grown overnight at 37 1C in Luria-
Bertani medium (LB, 10 g of tryptone and 5 g of yeast extract
per liter). Cells were harvested by centrifugation, washed twice
with phosphate-buffered saline (PBS, NaCl, 8.0 g Lꢁ1; KCl,
Scheme 1 Synthetic procedure of the imide N-halamine-loaded PMMA
nanoparticles based on 5-allylbarbituric acid.
0.20 g Lꢁ1; Na2HPO4ꢂ12H2O, 3.49 g Lꢁ1; KH2PO4, 0.2 g Lꢁ1
;
pH 7.4), and diluted to concentrations of 106–7 colony-forming
units per mL. 100 mg of each sample was dispersed in 0.45 mL
sterilized distilled water, vortexed, and then sonicated for
30 min. In the antibacterial test, 50 mL of bacteria suspension and
450 mL of sample suspension were well mixed, and the mixture was
incubated under constant shaking. After a certain period of contact
time, 4.5 mL of 0.03 wt% sodium thiosulfate aqueous solution,
which was sterilized by passing through 0.22 mm membrane and
exhibited no effect on the growth of bacteria, was added into the
reaction suspension to neutralize the active chlorine and stop the
antibacterial action of the sample. The resulting mixture was mixed
well, serially diluted, and then 100 mL of each dilution was dispersed
onto LB agar plates. Colonies on the plates were counted after
incubation at 37 1C for 24 h.
3. Results and discussion
3.1 Characterization of poly(ABBA-co-MMA) nanoparticles
The fabrication of imide N-halamine-loaded PMMA nano-
particles mainly involves three steps, and each step is well
controllable. As shown in Scheme 1, 5-allylbarbituric acid was
synthesized from diethyl malonate by the cyclization reaction
between urea and diethyl allylmalonate, and copolymerized
subsequently with methyl methacrylate via radical polymeriza-
1
tion followed by chlorination. H NMR spectra of ABBA, MMA,
and poly(ABBA-co-MMA) are illustrated in Fig. 1. The measure-
ment was carried out with the aid of DMSO solvent, and the
corresponding fitting signal for DMSO was detected at d =
Fig. 1 1H NMR spectra of ABBA (A), MMA (B), and poly(ABBA-co-MMA) (C).
2.5 ppm, which is obviously observed in all three spectra.40 (Fig. 1B), the CH2QC group shows signals at d = 5.8 ppm and
The assignments of the signals in ABBA are d = 2.8, 3.7, 5.1, and 6.0 ppm, and the C–CH3 and O–CH3 displayed the resonance peak
5.6 ppm, respectively in Fig. 1A. In the spectrum of MMA at d = 3.8 ppm and 1.8 ppm, respectively. After copolymerization,
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Fig. 3 TEM image of the imide N-halamine-loaded PMMA nanoparticles
(A) and pure PMMA nanoparticles (B). The inset in (A) is the magnified TEM
image of the imide N-halamine-loaded PMMA nanoparticles.
spherical, smooth, and solid. The particle sizes are in a narrow
range of 470–600 nm with an average size of B522 nm. The
magnified TEM image of the imide N-halamine-loaded PMMA
nanoparticles is given in the inset of Fig. 3A to further verify the
Fig. 2 FTIR spectra of ABBA (A), MMA (B), and poly(ABBA-co-MMA) (C).
CH-, CH2-, and CH3- signals for poly(ABBA-co-MMA) were clearly surface state, showing a flawless surface without any cracks or
observed in Fig. 1C,41,42 whereas the CH2QC signals of the ABBA degradation. To substantiate the effect of the introduction of
and MMA components can no longer be detected. The corres- N-halamine on the microstructure, pure PMMA nanoparticles with
ponding peaks in the 1H NMR spectra reflected the successful similar diameter were also synthesized byradical polymerization
formation of poly(ABBA-co-MMA) via radical copolymerization of and characterized by TEM as shown in Fig. 3B. No significant
ABBA with MMA. Introducing MMA as a comonomer can not only difference is observed in the microstructure between pure PMMA
skillfully avoid the autoinhibition effect of ABBA but also enhance and their N-halamine modified counterparts. This reveals that
the reactivity of ABBA towards the chain propagation reaction.
the morphology, structure, and surface state of the as-synthesized
FTIR analysis was also performed to further confirm the nanoparticles were not influenced by introducing the N-halamine
existence of the functional groups of the different samples. component.
Fig. 2 shows the FTIR spectra of ABBA (A), MMA (B), and
Detailed information about the chemical composition of the
poly(ABBA-co-MMA) (C). The peaks at around 2985, 2926, and poly(ABBA-co-MMA) nanoparticles before and after chlorination was
1435 cmꢁ1 are assigned to the stretching vibration and bending provided by XPS measurement as shown in Fig. 4. XPS is used
vibration of the C–H bond, respectively.43 The characteristic to assess the surface composition, and the detection depth for
strong peak at around 1726 cmꢁ1 corresponds to the vibration inorganic materials is about 2 nm and less than 10 nm for organic
of the CQO bond.44 These peaks mentioned above are observed materials.49 The characteristic peaks assigned to photoelectrons
in all three spectra. In Fig. 2A, the peak at 1248 cmꢁ1 corresponds originating from the C 1s, N 1s, and O 1s energy level appear for
to the C–N stretching vibration, and the N–H stretching peak both samples at 285, 400, and 533 eV, respectively, which act as the
appears at 3200–3400 cm .
ꢁ1 45 The band at 1639 cmꢁ1 refers to the signal markers for the ABBA and MMA components.50 In particular,
stretching mode of the CQC bond.46 In Fig. 2B, besides the CQC the presence of the N 1s characteristic peak magnified in the inset
band, the peak at 1093 cmꢁ1 is attributed to C–O–C stretching image further reflects the successful immobilization of the barbi-
vibration.47 After copolymerization, the characteristic peaks turic acid group on PMMA nanoparticles. The significant difference
corresponding to C–N, N–H, and C–O–C bonds were also observed in the Cl 2p peak between poly(ABBA-co-MMA) (Fig. 4A) and imide
for poly(ABBA-co-MMA) in Fig. 2C, while the CQC stretching peak N-halamine-loaded PMMA nanoparticles (Fig. 4C) is clearly seen
disappeared at 1639 cmꢁ1, further demonstrating the fracture of from the magnified inset images, suggesting the formation of an
the CQC bond during the radical copolymerization.
N–Cl bond after chlorination treatment. Two weak peaks corres-
ponding to the photoelectrons from Si 2s and Si 2p energy level are
obtained at 154 and 103 eV possibly from the Si substrate used for
sample immobilization.51 For further clarification, the chemical
3.2 Characterization of imide N-halamine-loaded PMMA
nanoparticles
The fabrication of imide N-halamine-loaded PMMA nanoparticles bonds of the samples are identified by deconvolution of the C 1s
was accomplished by chlorination of poly(ABBA-co-MMA) nano- peak from the total XPS spectrum. All chemical bonds referred to C
particles. TEM acts as an effective tool for microstructure character- element can be captured by the deconvolution of the C 1s peak. One
ization and was carried out to investigate the morphology, structure, can see that both peaks’ fitting of C 1s (Fig. 4B and D) have been
surface state, and particle size of the products.48 Fig. 3A presents the curved into C–C, C–N, C–O, and CQO peak components.52 It is
typical TEM images of the imide N-halamine-loaded PMMA nano- quite reasonable to conclude from the C–O and C–N peak that MMA
particles. Except for a tiny minority that are conglutinate and and ABBA exist either at or very near the particle surface. Generally,
tortuous, most nanoparticles are uniform, quasi-monodisperse, the presence of C 1s, O 1s, N 1s, and Cl 2p peak in the spectra not
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Fig. 4 XPS survey (A and C) and C 1s (B and D) spectra of poly(ABBA-co-MMA) (A and B) and imide N-halamine-loaded PMMA nanoparticles (C and D).
only exhibits the surface chemistry but also confirms the formation be utilized as bactericides as illustrated in Scheme 2. In this
of imide N-halamine-loaded PMMA nanoparticles.
study, the poly(ABBA-co-MMA) nanoparticles were immersed in
sodium hypochlorite solution, and the chlorine content of the
as-synthesized imide N-halamine-loaded PMMA nanoparticles
was determined by an iodometric/thiosulfate titration method.54
It can be expected that imide N-halamine-loaded PMMA nano-
particles have potent antibacterial capabilities arising from the
N-halamine component. A zone of inhibition study is an effective
3.3 Antimicrobial assessment
The N–H structure can be transformed facilely into an N–Cl
group upon treatment with sodium hypochlorite to obtain the
target N-halamine product.53 The as-synthesized products can
Scheme 2 Picture showing microorganism treated with the imide N-halamine-loaded PMMA nanoparticles.
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approach for determining the antimicrobial behavior of bio-
cides.55 It is well accepted that the zone of inhibition is propor-
tional to the bactericidal activity. The larger the diameter of the
inhibition zone (DIZ), the more active the bactericide. Herein,
the DIZ value of the imide N-halamine-loaded PMMA nano-
particles was assessed using E. coli as the representative micro-
organism by a disk diffusion test. For comparison, the DIZ value
of pure PMMA was estimated as well. The control experiments
have a robust growth of bacteria. Pure PMMA nanoparticles do
not show any inhibition zone, indicating that the support
component is not toxic to the bacteria. Unlike pure PMMA, the
imide N-halamine-loaded PMMA nanoparticles show a clear
inhibition ring size of 5.6 mm, indicating significant antibacterial
activity against E. coli. Accordingly, it is considered that the
excellent biocidal function of the imide N-halamine-loaded
PMMA nanoparticles is provided completely by the N-halamine
component.
The antibacterial performance of the imide N-halamine-
loaded PMMA nanoparticles was further examined against
P. aeruginosa by using the spread plate technique.56 Similarly,
the antibacterial assay of the pure PMMA nanoparticles was
carried out comparatively by the same method. No significant
difference is found between the control and PMMA nano-
particles, whereas an obvious decrease is detected in the
population of the bacterial colonies after exposure to the imide
N-halamine-loaded PMMA nanoparticles. This is in good agree-
ment with the DIZ results. The excellent disinfectant behavior
of the imide N-halamine-loaded PMMA nanoparticles based on
barbituric acid makes them promising candidates for deacti-
vating bacteria or even for disease control.
Fig. 5 Antibacterial kinetic test graphs for pure PMMA and the imide
N-halamine-loaded PMMA nanoparticles against P. aeruginosa (A) and
S. aureus (B).
3.4 Effect of contact period
The antimicrobial kinetic test confirmed the effect of contact
time on the biocidal capability of imide N-halamine-loaded
PMMA nanoparticles. The antibacterial kinetic curves, shown may be attributed to the different cell structures of P. aeruginosa
in Fig. 5, present the biocidal activity of pristine PMMA and and S. aureus.58
their imide N-halamine-loaded counterparts by selecting both
P. aeruginosa and S. aureus. The number of surviving bacterial
3.5 Effect of N-halamine structure
colonies was counted as a function of the contact time from It is acknowledged that the bactericidal activity of N-halamine
0 min to 60 min. As expected, PMMA nanoparticles almost is in the order imide 4 amide 4 amine. In our previous
show a horizontal development trend, whereas significant reports, we designed several hydantoin-based N-halamine
bacterial reduction is observed upon exposure to imide nanoparticles and their antimicrobial behavior was studied
N-halamine-loaded counterparts with both P. aeruginosa and systematically. Hydantoins contain one imide and one amide
S. aureus for the whole contact time range. A sharp increasing group in their structure, while barbituric acid is a heterocyclic
trend in bacterial reduction is observed within the initial compound with two imide groups. Therefore, it is reasonable to
contact time range, and bactericidal speed slows down gradu- consider that barbituric acid-type N-halamine would be more
ally as the exposure period is extended, which suggests that the effective than hydantoin-based N-halamine for deactivating
imide N-halamine-loaded PMMA nanoparticles are more suita- bacteria. To substantiate this hypothesis, the bactericidal
ble for fast sterilization.18,30,57 The antibacterial results also capability of barbituric acid-originated N-halamine nanoparticles
indicated that the imide N-halamine-loaded PMMA nano- was quantitatively compared with hydantoin-structural N-halamine
particles have different capabilities towards P. aeruginosa and nanoparticles via the test of minimum inhibitory concentration
S. aureus. As a general observation, the products provided faster (MIC). MIC was defined as the sample concentration at which the
antibacterial action against P. aeruginosa than against S. aureus colonies were reduced in the CFU per mL numbers of Z3 log.59
for the same contact time. Therefore, it can be considered that Herein, the MIC value of the samples against S. aureus and
P. aeruginosa would be more vulnerable than S. aureus towards P. aeruginosa was determined by the agar plate method as
the N-halamine-based antibacterial agents. This phenomenon shown in Fig. 6. The MIC values of barbituric acid-based
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Table 1 Reduction of bacterial colonies (S. aureus) after 60 min exposure
to the imide N-halamine-loaded PMMA nanoparticles with different par-
ticle size and activated surface area
Sample
Size (nm)
Surface area (m2 gꢁ1
)
Reduction (%)
S1
S2
S3
S4
523.81
809.52
904.76
976.19
11.45
7.41
6.63
6.15
95.63
80.37
73.36
55.12
representative SEM image of imide N-halamine-loaded PMMA
nanoparticles with different diameters. Except for particle size,
the morphology, structure, and surface state show no distin-
guishable difference among them.
Fig. 6 Minimum inhibitory concentration (MIC) of hydantoin-originated
(A) and barbituric acid-based (B) imide N-halamine-loaded PMMA nano-
particles towards S. aureus and P. aeruginosa.
Normally, the particle size is inversely proportional to the
surface area. The corresponding surface area of the products
with different size was calculated based on the assumption that
the nanoparticles are non-porous spheres with density of
1.0 g cmꢁ3. The calculation was performed according to the
following equation: S = 6(Dꢂd)ꢁ1, wherein S is the surface area
N-halamine nanoparticles against S. aureus and P. aeruginosa
are 40 and 20 mg mLꢁ1, respectively, and 80 and 80 mg mLꢁ1
for hydantoin-based N-halamine nanoparticles. It is well known
that the MIC value of a biocide reflects the magnitude of
the susceptibility to the bacteria, and the antibacterial capability is
inversely proportional to the MIC value. Thereby, the antibacterial
efficiency of barbituric acid-based N-halamine nanoparticles is
higher than that of hydantoin-based counterparts. This pheno-
menon is attributed to the structure difference between barbituric
acid-based and hydantoin-originated N-halamine.30,32
(m2 gꢁ1); D is the diameter (mm); and d is the density (g cmꢁ3
)
of the sample.60 The antimicrobial efficiency of the imide
N-halamine-loaded PMMA nanoparticles with different size
against S. aureus was determined comparatively. Table 1 sum-
marizes the trend of bactericidal activity correlated to particle
size and surface area within the same contact time of 60 min.
Nanoparticles with particle size of 523.81 nm and surface area
of 11.45 m2 gꢁ1 show as high as 95.63% colonial reduction, and
the antimicrobial efficiency decreases with increasing particle
size/decreasing surface area, and the 976.19 nm and 6.15 m2 gꢁ1
nanoparticles can only kill 55.12% of the S. aureus. In general,
the imide N-halamine-loaded PMMA nanoparticles with smaller
size/higher surface area have stronger bactericidal capability towards
bacterial colonies. The performance of the imide N-halamine-
loaded PMMA nanoparticles with different diameter proves the
size effect hypothesis.
3.6 Effect of particle size
As for N-halamines, the particle size is a decisive parameter
determining the bactericidal capability. The antimicrobial effi-
ciency can be enhanced by reducing the particle size because
the expanded surface area provides more activated surface sites
that can kill the bacteria. To confirm the size effect, imide
N-halamine-loaded PMMA nanoparticles with different particle
sizes (523.81 nm, 809.52 nm, 904.76 nm, and 976.19 nm)
were prepared and characterized by TEM. Fig. 7 illustrates the
3.7 Effect of chlorine content
Chlorine content also plays an important role in governing
the antibacterial activity of N-halamines.61 The relationship
between the chlorine content and biocidal activity is con-
structed by means of S. aureus reduction as a function of
chlorine content as shown in Fig. 8. In general, bacterial
reduction displays an upward trend with increasing chlorine
loading. Merely 51% S. aureus reduction is observed for the
0.07 Cl% sample, while as high as 95% reduction is seen for the
0.61 Cl% sample after 60 min contact with the biocides.
Consequently, it is clear that the antimicrobial capability of
N-halamines is proportional to the chlorine content, because
the product with higher chlorine loading can offer more
activated sites, resulting in the enhanced antibacterial effi-
ciency. Interestingly, bacterial colonies reduce drastically with
increasing chlorine content within the lower chlorine content
region, and then level off with extending the chlorine loading.
The most probable reason is detailed as follows. Although
increased chlorine content usually leads to enhanced bactericidal
Fig. 7 SEM image of the imide N-halamine-loaded PMMA nanoparticles
with different particle size.
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barbituric acid-based and hydantoin-based N-halamine. Bacteri-
cidal capability of the imide N-halamine-loaded poly(methyl
methacrylate) nanoparticles with different particle sizes and
different chlorine contents was studied systematically as well.
After long-term storage, the imide N-halamine-loaded poly(methyl
methacrylate) nanoparticles showed excellent stability in the dry
state. We believe that the present study opens up the possibility
for extensive investigation of the antimicrobial N-halamines,
broadening their practical applications in various fields.
Acknowledgements
This research was supported by the National Natural Science
Foundation of China (21304044) and the Supported by Program
of Higher-level Talents of Inner Mongolia University (30105-125136).
Fig. 8 Reduction of bacterial colonies (S. aureus) after 60 min exposure
to the imide N-halamine-loaded PMMA nanoparticles with different
chlorine content.
Notes and references
efficacy, it can also render the surface more hydrophobic, resulting
in poorer contact with the bacteria and thus less efficacy. Gen-
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