Silver nanoparticles with pH induced surface charge switchable properties for antibacterial and antibiofilm applications

Article abstract of DOI:10.1039/c8tb02917b

Silver nanoparticles (AgNPs) are widely used as antibacterial agents because of their significant antimicrobial activities and little sign of antimicrobial resistance. However, the relatively high toxicity to healthy cells and low penetration efficiency into bacterial biofilms prevent their further use in biomedical applications. In order to decrease the cytotoxicity of the AgNPs to mammalian cells while increasing their antibacterial and antibiofilm efficiency, a novel nanocomposite composed of AgNPs decorated with carboxyl betaine groups (AgNPs-LA-OB) was prepared. The zwitterion modified AgNPs showed a pH responsive transition from a negative charge to a positive charge, which enabled the AgNPs to be compatible with mammalian cells and red blood cells (RBCs) in healthy tissues (pH ～ 7.4), while strongly adhering quickly to negatively charged bacterial surfaces at infectious sites (pH ～ 5.5) based on electrostatic attraction. The AgNPs penetrated deeply into bacterial biofilms and killed the bacteria living in an acidic environment. The results indicated that the designed zwitterion NPs for antibacterial applications and eradication of bacterial biofilms, which also had particles that did not harm the healthy cells showed promise for future use in humans. The satisfactory selectivity for bacteria compared to RBCs, together with their potent eradication of bacterial biofilms make AgNPs-LA-OB a promising antibacterial nanomedicine in biomedical fields.

Full text of DOI:10.1039/c8tb02917b

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Materials Chemistry B
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Silver nanoparticles with pH induced surface
charge switchable properties for antibacterial
Cite this:DOI: 10.1039/c8tb02917b
and antibiofilm applications†
Zhuangzhuang Qiao, Yan Yao, Shaomin Song, Meihui Yin and Jianbin Luo
*
Silver nanoparticles (AgNPs) are widely used as antibacterial agents because of their significant
antimicrobial activities and little sign of antimicrobial resistance. However, the relatively high toxicity to
healthy cells and low penetration efficiency into bacterial biofilms prevent their further use in biomedical
applications. In order to decrease the cytotoxicity of the AgNPs to mammalian cells while increasing
their antibacterial and antibiofilm efficiency, a novel nanocomposite composed of AgNPs decorated with
carboxyl betaine groups (AgNPs-LA-OB) was prepared. The zwitterion modified AgNPs showed a pH
responsive transition from a negative charge to a positive charge, which enabled the AgNPs to be
compatible with mammalian cells and red blood cells (RBCs) in healthy tissues (pH B 7.4), while strongly
adhering quickly to negatively charged bacterial surfaces at infectious sites (pH B 5.5) based on
electrostatic attraction. The AgNPs penetrated deeply into bacterial biofilms and killed the bacteria living
in an acidic environment. The results indicated that the designed zwitterion NPs for antibacterial
applications and eradication of bacterial biofilms, which also had particles that did not harm the healthy
cells showed promise for future use in humans. The satisfactory selectivity for bacteria compared to
RBCs, together with their potent eradication of bacterial biofilms make AgNPs-LA-OB a promising
antibacterial nanomedicine in biomedical fields.
Received 6th November 2018,
Accepted 26th December 2018
DOI: 10.1039/c8tb02917b
rsc.li/materials-b
NPs to microorganisms can also be active against mammalian
cells. Generally, the relatively high toxic effects to human cells and
1. Introduction
With the increasing threat of microbial infections, especially in low yield for penetration through the bacterial biofilms of AgNPs
hospitals and for community acquired infections, there is a are the two main obstacles for their biomedical applications.24
desperate need to design and develop highly effective treatment
Polymers or surfactants were coated on AgNPs to stabilize
modalities to inhibit the infections.1–4 Antibiotics have been and/or to reduce its toxicity to mammalian cells. For example,
shown to be effective in combating pathogenic microbes and AgNPs decorated on lipase-sensitive polyurethane micelles
have saved numerous lives in the past century.5–7 Unfortunately, (Ag-PUM) were recently reported, which showed relatively low
frequent and excessive use of conventional antibiotics has caused cytotoxicity because of the aggregation of small AgNPs into
extensive multidrug resistance in bacterial infections.8–12 Thus, it PUMs with dense poly(ethylene glycol) brushes.25,26 The release
is very necessary to develop effective and safe antibacterial of small AgNPs in the presence of lipase resulted in excellent
agents.13–16 Silver nanoparticles (AgNPs) have been known to antibacterial activities of AgNPs. An ultrathin layer (1–2 nm) of
exhibit powerful antimicrobial activities and have been widely silver was coated onto a magnetic core with a ligand gap was
applied in medicine because of their high antimicrobial activity prepared by Webster et al. to increase the penetration of AgNPs
and relatively good biocompatibility.17–23 However, the cytotoxic into bacterial biofilms when an external magnetic field is
effects and the bactericidal activities of AgNPs remain two applied.27 A ultrathin layer of gold was further coated onto the
principal contradictions because the larger surface area and silver rings using another ligand gap to improve the cytotoxicity of
smaller size of the NPs are expected to improve the antibacterial AgNPs which also gave the NPs with a photothermal bactericidal
activity; meanwhile, the mechanisms involved in the toxicity of effect.24
Most bacterial membranes are negatively charged whereas
the mammalian cells are of negligible net charge. In addition,
College of Chemistry and Environmental Protection Engineering,
an acidic microenvironment is always found in bacterial infections
Southwest Minzu University, Chengdu 610041, China. E-mail: luojb1971@163.com;
and in bacterial biofilms.28–30 Taking these factors into account,
Tel: +86 28 85522269
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8tb02917b pH induced charge switchable AgNPs were prepared to decrease
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Scheme 1 Silver nanoparticles with charge switchable properties and their interaction with mammalian cells and bacteria.
their cytotoxicity towards mammalian cells in neutral solutions calcium hydride and distilled before use. N,N-Dimethylethanol-
while increasing their toxicity to bacteria in an acidic micro- amine (DMEA, Keshi, Chengdu), chloroacetic acid (J&K Scientific,
environment. To achieve this, carboxyl betaine (CB) groups were Beijing), trypticase soy agar medium (TSA, AOBOX, Hengshui),
covalently bonded on the surface of AgNPs using disulfide trypticase soy liquid medium (TSB, AOBOX, Hengshui) were
bonds. As shown in Scheme 1, the carboxyl groups deprotonated used as received. Dialysis tubing with a molecular weight cutoff
at physiological conditions which resulted in AgNPs with zwit- of 200 and 1000 was purchased from Sinopharm Chemical
terions and which showed good cytocompatibilities. However, Reagent (Shanghai, China).
the protonation of carboxyl groups in an acidic fluid results in
positively charged AgNPs, which enhance the interaction of NPs
and bacteria. As far as is known, AgNPs with pH induced surface
charge transform activities with an enhanced antibacterial efficiency
and reduced cytotoxicity have not been reported previously.
The biofilm is a layer of bacterial cells adhered to the surface
of a material or tissue with self-produced extracellular polymeric
substances (EPS). The EPS serves as a protective barrier against
antibiotic infiltration and cellular attack by the host’s innate
immune cells. It is reported that positively charged NPs show
better penetration through bacterial biofilms because of the
negative charge nature of EPS.31–34 In general, the metabolic
activity of bacteria is very active and diversified, and its rapid
propagation is a notable feature, which causes a decrease in the
local environment pH to occur at the infection site. The biofilm
environment is also inconsistent, usually closer to the matrix,
with a lower oxygen concentration and pH value. Surface charge
adaptive gold nanoparticles or polymer micelles (capsules) have
been shown to have good penetration through bacterial biofilms
by taking advantage of the pH difference between the physiological
environment and the bacterial biofilms.35–37 Based on these
results, it is expected that a silver-zwitterion nanocomposite will
achieve better penetration through bacterial biofilms and kill the
bacteria embedded in the biofilms.
2.2 Characterization
Proton nuclear magnetic resonance 1H-NMR spectra were obtained
using a Mercury Plus 400 (Varian) NMR spectrometer at room
temperature with deuterium oxide (D2O) or deuterated dimethyl-
sulfoxide (DMSO-d6) as a solvent. The chemical shifts were reported
in ppm relative to tetramethylsilane.
Zeta potential studies of the aqueous solution of AgNPs were
carried out using a Nano-ZS 90 Nanosizer (Malvern Instruments
Ltd, UK) at various pH values. No background electrolyte was
added. Each measurement was repeated three times.
Ultraviolet-visible (UV-vis) studies were conducted using a
UnicamUA500 UV-vis spectrophotometer (Thermo Electron
Corporation) with a scan speed of 300 nm minꢀ1. The absorbance
and transmittance spectra of the AgNPs were recorded in the range
of 300–600 nm.
Inductively coupled plasma-mass spectrometry (ICP-MS)
studies of the AgNPs were carried out using an Agilent 7700 ce
instrument (Agilent Technologies).
Scanning electron microscope (SEM) images were taken using
an Inspect F field emission scanning electron microscope
(FE-SEM, FEI, USA) at 20 kV.
Transmission electron microscopy (TEM) was carried out
using a Tecnai G2 F20 transmission electron microscope (Royal
Philips Electronics Ltd, The Netherlands) at an acceleration
voltage of 200 kV.
2. Experimental
2.1 Materials
2.3 The synthesis of oxibetaine (OB) and lipoic acid anhydride
(LA)
Silver nitrate (AgNO3), N,N0-dicyclohexylcarbodiimide (DCC,
99%), 4-(dimethylamino)pyridine (4-DMAP, 99%), thioctic acid In a three-necked flask, DMEA (50 mmol, 4.46 g) was dissolved
(TA, 98%) and sodium borohydride (NaBH4) were obtained in anhydrous alcohol, followed by dropwise addition of chloroacetic
from Sigma-Aldrich. Dichloromethane (DCM) was dried over acid (60 mmol, 5.67 g) which was dissolved in anhydrous alcohol at
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Table 1 Feed ratios for the preparation of LA-OB coated AgNPs
80 1C under vigorous stirring. The reaction was allowed to proceed
under refluxing conditions for 12 h to ensure that the complete
reaction had occurred. The mixture was then cooled to room
temperature, followed by the addition of 500 mL of ethyl ether to
start the precipitation. The crude OB was purified three times by
dissolving it in anhydrous alcohol and precipitating it in ethyl ether.
The residual solvent in the product was removed under vacuum for
several hours to obtain pure OB.
Mass (mg)
LA-OB
Sample
AgNO3
AgNPs–LA-OB 1 : 0.05
AgNPs–LA-OB 1 : 0.1
AgNPs–LA-OB 1 : 0.25
AgNPs–LA-OB 1 : 0.5
50
50
50
50
2.5
5
12.5
25
Lipoic acid anhydride was synthesized according to a procedure
reported elsewhere with slight modification.38–41 Briefly, TA
(5 mmol, 1.03 g) was dissolved in anhydrous DCM (10 mL), and
DCC (3 mmol, 0.65 g) was dissolved in DCM (5 mL) at room
temperature. Subsequently, the precipitate formed in the solution
which was filtered to remove dicyclohexylurea. Infrared spectro-
scopy was used to examine the filtrate for the presence of LA
(1735 and 1805 cmꢀ1) and the absence of the parent carboxylic
acid (1701 cmꢀ1).
AgNPs (34.74, 17.37, 8.68, 4.34, 2.17, 1.09, 0.55 mg mLꢀ1). Then
40 mL of bacterial solution was added and the suspensions were
shaken on a shaker at 200 rpm at 37 1C. Diluted broth containing
the bacterial suspension without AgNPs-LA-OB was used as the
control samples.
The minimum bactericidal concentration (MBC) of an anti-
bacterial agent were also measured and used for the evaluation
of the antibacterial effects of AgNPs. Briefly, 100 mL of AgNPs-
LA-OB solution was put in each zero dilution well of polystyrene
96-well plates. Adjusted bacterial suspension (50 mL) was put
into each zero dilution well of a preset microplate, to achieve
5 ꢁ 105 colony forming units (CFU) mLꢀ1 in each well (150 mL).
Bacteria were allowed to grow aerobically at 37 1C for 18 h.
Subsequently, a culture suspension of 100 mL from each well
was uniformly coated on plates. After overnight incubation at 37 1C,
bacterial colonies became visible and the lowest concentration at
which the colony formation remained absent was taken as the MBC.
The experiments were repeated at least three times.
2.4 Synthesis of the ligand of 2-[5-(1,2-dithiolan-3-yl)
pentanoyloxy]-N-(carboxymethyl)-N,N-dimethylethanaminium
(LA-OB)
The synthesis procedure was carried out using a standard
method under nitrogen protection. Briefly, an OB solution
(0.028 g, 0.19 mmol) in ethyl alcohol (5.0 mL) was added to a
4-DMAP (46 mg, 0.38 mmol) solution which was dissolved in
DCM (1.0 mL), and LA (0.15 g, 0.38 mmol) in DCM (1.0 mL) was
then added dropwise. The mixture was stirred for 48 h at room
temperature. The final product was isolated by precipitation in
cold diethyl ether, washed several times with diethyl ether and
then dried under a vacuum at room temperature.
2.7 Biofilm culture and treatment
The biofilms of S. aureus were formed on Fisherbrand cover
glass slides (Sanger Biotechnology Co., Ltd) according to a
method published in the literature.45,46 The S. aureus strains
were grown aerobically in TSB broth overnight at 37 1C and the
2.5 Preparation of silver nanoparticles surface coated with
carboxyl betaine (CB) groups (AgNPs-LA-OB)
Silver nanoparticles with pH induced surface charge switchable resulting bacterial suspensions were then serially diluted in
properties were synthesized using the reduction of AgNO3 in TSB and adjusted to a cell density of 106 CFU mLꢀ1. Then, 1 mL
the presence of LA-OB. At first, 0.05 g LA-OB was dissolved in of S. aureus suspension was seeded into each well on which was
10 mL of water (H2O) which was then mixed with 5 mL of placed a Fisher brand cover glass slide to allow the biofilm
AgNO3 solution in a 20 mL sample bottle. Subsequently, NaBH4 formation for seven days at 37 1C. The culture medium was
solution (2 mL) was added dropwise to the prepared solution refreshed every day.
under vigorous stirring. The specific composition of each type
Biofilm treatment was quantified in triplicate using a modified
of AgNP is listed in Table 1. After 12 h of reduction, the solution procedure that had been reported in the literature.47–50 Briefly,
was further purified using dialysis against H2O for 48 h. The the mature biofilm of S. aureus growing on the surface of the
concentration of silver in the purified solutions was determined coverglass was gently rinsed three times with sterile phosphate
using ICP-MS measurement.
buffered saline (PBS) to remove the non-adhering bacteria and
then 200 mL (8.68 mg mLꢀ1) of AgNP–LA-OB suspension with a
pH of 7.4 or 5.5 were slowly added. The control group was
treated with 200 mL of 0.01 M PBS as substitute for the treatment.
2.6 Antibacterial activities against Escherichia coli and
Staphylococcus aureus
Two bacterial strains, S. aureus (ATCC 29213) and E. coli (ATCC After incubation for 16 h contact time, the suspension was
35218), were used in this experiment. The antibacterial activities removed. A standard plate counting assay was used to assess
of the AgNPs-LA-OB were evaluated using minimal inhibitory the vitality of the residual bacteria within the biofilms. The
concentration (MIC) and optical density (OD) readings at a treated biofilms were carefully rinsed three times with sterile
wavelength of 600 nm of the microorganism solutions, which PBS to remove the planktonic bacteria and then re-suspended in
were measured every 2 h from time 0 h to 24 h using UV-vis individual 10 mL polyethylene tubes containing 2 mL of physio-
spectrometry.42–44 Firstly, the AgNPs-LA-OB with an initial silver logical sterile solution were then ultrasonically mixed intermittently
concentration of 69.47 mg mLꢀ1 was sterilized under UV light for to detach and disaggregate the biofilm. Subsequently, the resulting
2 h and diluted gradually to obtain several concentrations of suspensions were serially diluted and 100 mL diluted samples
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from each tube were used to coat the plates. The plates were (5) Control group: the bacteria (1.0 mL) in another centrifuge tube
incubated at 37 1C for 16 h and the number of bacteria were without the addition of antibacterial NP solution were shaken well
then counted. A LIVE/DEAD BacLight Bacterial Viability kit and then cultivated on a shaking bed at 37 1C for 8 h, followed by
(L-7012, Invitrogen) was used to further visually examine the centrifugation at 5000 rpm for 30 min. The supernatant was
anti-biofilm activities of the AgNPs-LA-OB. They were then removed and 2.0 mL of PBS was poured into the centrifuge tube.
incubated with 30 mL (3 mM) propidium iodide and SYTO-9 The tube was shaken well and centrifuged for 30 min, and then the
green fluorescent nucleic acid stain for 30 min in darkness at residual supernatant was removed. (6) Immobilization of bacterial
room temperature. Finally, the penetration of AgNPs-LA-OB, morphology. Phosphate buffer (pH 7.4; 0.5 mL) containing 2.5%
under different pH with different surface charges, into biofilms glutaraldehyde was added to two centrifuge tubes of the experi-
were examined using confocal laser scanning microscopy (CLSM; mental and control groups. The tubes were shaken well and then
Olympus FV1000, Japan). Each test was carried out in triplicate. placed in the fridge overnight at 4 1C to fix the bacterial
morphology. (7) TEM sample preparation. The two tubes were
2.8 SEM observation of biofilm treated with silver
nanoparticles
centrifuged for 30 min and the supernatants were removed. Then
sterile water was added into both tubes. The tubes were shaken
SEM was used to visually examine the biofilm surface topography well and centrifuged for 30 min. After the removal of the super
with or without the presence AgNP–LA-OB for 2 h or 16 h supernatant, approximately 0.5 mL of sterile water was added to
according to a modified protocol.51–53 Briefly, Fisherbrand cover both tubes. These bacterial suspensions were then shaken well
glass slides covered with biofilms were carefully washed three and were then ready for preparation for TEM observation.
times with sterilized PBS and then 200 mL (8.68 mg mLꢀ1) of
2.10 Hemolysis assay
AgNP–LA-OB suspension in a neutral pH of 7.4 or an acidic pH of
5.5 or 4.7 environment were slowly added. The control groups
were treated with 200 mL of 0.01 M PBS as substitute for the
treatment. After treatment, the suspension was discarded and
washed in PBS, and then fixed in 2.5% (v/v) glutaraldehyde at 4 1C
for 4 h. The coverslip covered with biofilms was then dehydrated
in ethanol water mixtures with increasing ethanol concentrations
(50%, 70%, 85%, 90%, 95% and 100%). Finally, the biofilms were
dried and examined using FE-SEM.
All the experimental protocols used followed the guidelines of
the Animal Care and Use Committee, Southwest University for
Nationalities and the experiments were approved by the Committee.
Healthy Kunming mice were obtained from Chengdu Dossy
Experimental Animals (Chengdu, Sichuan, China). The hemo-
lysis assays were conducted using heparin stabilized red blood
cells (RBCs), which were freshly collected from the mice.56–58
Briefly, fresh mouse whole blood was washed with PBS solution
and isolated using centrifugation at 1500 rpm for 10 min.
Following the last wash, the RBCs were diluted in PBS to achieve
8% blood content (by volume). The diluted RBC suspension
(200 mL) was mixed with different concentrations of NP solution
for a 2 h incubation period. After centrifugation to isolate the
RBCs, the supernatant sample was transferred to a quartz colori-
metric utensil and the spectrum was recorded using a UV-vis
spectrophotometer at 570 nm. A negative control solution that
contained only PBS was used as a reference for 0% hemolysis. Pure
deionized water was used as positive control for hemolysis test.
The percentage hemolysis of the RBCs was calculated using the
following equation: percentage hemolysis = [(sample absorbance ꢀ
negative control absorbance)/(positive control absorbance ꢀ
negative control absorbance)] ꢁ 100. The test was repeated
three times for each sample.
2.9 TEM study of the interaction between bacteria and AgNPs
To study of the interaction between the bacteria and the AgNPs,
TEM was used to observe the morphologies of the micro-
organisms before and after treatment with AgNPs. The detailed
procedure can be found in the literature elsewhere.54,55 The
brief methodology is: (1) the bacteria were activated by cultivating
aerobically in lysogeny broth (LB) broth at 37 1C for 24 h and
the activated bacteria in LB broth (5.0 mL) were centrifuged
at 5000 rpm for 20 min and the supernatant was removed.
(2) Then 2.0 mL of broth containing bacteria were added into
the contents of the centrifuge tube again and the addition/
centrifugation/removal processes were repeated. Finally, 6.0 mL
of the bacterial strain was extracted. (3) Purification of bacteria.
Normal saline (2.0 mL) was poured into the centrifuge tube and
the residual supernatant was washed out after shaking the
tube, centrifuging for 30 min and removing the supernatant.
Then 2.0 mL of normal saline was added to the contents of the
centrifuge tube again. After shaking well, and the bacteria were
divided into two groups: experimental group (1.0 mL) and
3. Results and discussion
3.1 Preparation and characterization of AgNP–LA-OB
control group (1.0 mL). (4) Experimental group: the NP solution In this work, a convenient, efficient strategy for the synthesis of
(0.5 mL; 8.68 mg mLꢀ1) was added to the contents of the AgNPs with pH induced surface charge transform activities
centrifuge tube with 1.0 mL of bacteria. The mixture of bacteria (AgNP–LA-OB) was demonstrated. First the LA-OB were success-
and NPs was shaken well and then cultivated on a shaking bed fully synthesized using the three-step process illustrated in
1
at 37 1C for 8 h, followed by centrifugation at 5000 rpm for Scheme 2. After purification, the representative H-NMR spectra
30 min. The supernatant was removed and 2.0 mL of PBS was was obtained and the assignment of the peaks is presented in
poured into the centrifuge tube. The residual supernatant was Fig. 1. The characteristic signals of OB at B3.3 ppm, B3.9 ppm
then removed after shaking the tube and centrifuging for 30 min. and B4.6 ppm whereas the signals at B1.8 ppm and B3.22 ppm
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for further studies. The results of thermogravimetric analysis
indicated that the amount of LA-OB grafted on AgNPs was
about 38% (Fig. S1, ESI†).
The zeta potentials of the AgNP–LA-OB were determined
using dynamic light scattering at 25 1C with a Nano-ZS 90
Nanosizer. As shown in Fig. 2B, at physiological conditions or
above (pH 4 7.4), the AgNP–LA-OB exhibited a negative surface
charge because of the ionization of the carboxyl groups, suggesting
that there was a long circulation time in the blood stream.
However, the zeta potential of AgNPs increased as the pH value
decreased because of the protonation of the carboxyl groups,
which resulted in a positive surface charge of AgNPs in acidic
solutions (pH 5.0 or below). The exact silver concentration in
the prepared NPs dispersions was determined using ICP-MS
(Fig. S2, ESI†).
Scheme 2 Synthesis route of LA-OB.
3.2 Antibacterial activities against planktonic bacteria
Pathogenic bacteria usually present a negative surface charge
whereas mammalian cells show a negligible net surface charge.
In addition, an acidic microenvironment is always found in
bacterial infections and in bacterial biofilms. The AgNPs have
been shown to exhibit a powerful antimicrobial activity and
have been widely applied in medicine because of their high
antimicrobial activity and relatively good biocompatibility.
Although the antibacterial mechanism of the AgNPs has not been
definitively determined to date, AgNPs have a different mechanism
of killing bacteria compared with the quaternary ammonium salts
and other antibiotics. The results of some studies suggested that
the AgNPs interact with the cell membrane and some of them also
penetrate the bacterial cell wall, whereas others claimed that the
antimicrobial mechanism relies on the silver ions released by the
AgNPs or the formation of free radicals which induce damage to
the bacterial membrane thereby causing the death of bacteria.19 It
is well accepted that the bactericidal activity of AgNPs depends on
their size, shape, stability and their surface properties. In order to
Fig. 1 1H-NMR spectra of LA (A) in DMSO-d6 and OB (B), LA-OB (C) in
D2O.
were assigned to the two methylene protons in the lipoic ring, improve the stability and biocompatibility of AgNPs, previous
which indicated the successful synthesis of surface modification researchers have used surfactants and polymers to modify the
agents for AgNPs (LA-OB).
AgNPs.20 It is speculated that AgNPs with pH induced charge
Silver nanoparticles surface coated with CB groups were switchable activities can be used to decrease cytotoxicity towards
prepared by simply reducing the silver ions in the presence of mammalian cells in neutral solutions while increasing their
LA-OB. The disulfide groups of LA-OB were reduced to thiol toxicity against bacteria in an acidic microenvironment. More
which covalently attached the CB groups onto the AgNP surface importantly, the pH induced surface charge switchable AgNPs
with metal–sulfur bonds. The effect of the ratio of LA–OB : silver (AgNPs-LA-OB) penetrated into the bacterial biofilms and killed
on the size and morphology of the prepared AgNPs was studied the bacteria embedded in the biofilms because of the negative
by adding different amounts of AgNO3 to the reaction mixtures. nature of EPS and the acidic microenvironment of the biofilms.
The ratio of LA–OB : silver was varied from 1 : 0.05 to 1 : 0.5 as
As a proof-of-concept, the antibacterial activities of AgNPs-LA-
shown in Table 1. As is known, the optical properties were OB against Gram-negative (E. coli) and Gram-positive (S. aureus)
closely dependent to the size and morphological features of the bacteria were evaluated using MIC and minimum bactericidal
AgNPs. The UV-vis spectra were used to evaluate the resulting concentration (MBC) assay in a neutral or an acidic environment.
AgNPs-LA-OB prepared from different mass ratios of LA-OB The AgNPs-LA-OB were incubated with bacteria first and the
and AgNO3 and the results are shown in Fig. 2A and the TEM proliferation of the bacteria was then monitored during 24 h of
micrographs of the resulting nanocomposites are shown in cultivation on the basis of the ODs of the suspension measured
Fig. 2C. It is clearly shown from the UV-vis spectra and TEM at 600 nm (OD 600) every 2 h using a UV-vis spectrometer.
images that well distributed AgNPs with a diameter of several Additionally, the antimicrobial activities under a bacterial acidic
nanometers were obtained when the ratio of LA-OB and AgNO3 infection site (pH 5.5) or even lower, were determined. A buffer
was 1 : 0.1. Therefore, an AgNP–LA-OB ratio of 1 : 0.1 was used solution with a pH value of 5.5 or 4.7 was used to simulate the
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Fig. 2 (A) UV-vis absorption spectra, (B) zeta potential of AgNPs-LA-OB (1 : 0.1) at different pHs, (C) TEM micrographs of LA-OB coated AgNPs with
different mass ratios, (c1) 1 : 0.05, (c2) 1 : 0.1, (c3) 1 : 0.25, (c4) 1 : 0.5 and the corresponding micrographs.
Fig. 3 Dose-dependent growth inhibition of E. coli (A–C) and S. aureus (D–F) in the presence of various concentrations of AgNPs-LA-OB as a function
of time. Control: without AgNPs-LA-OB.
bacterial acidic infection tissue. The antibacterial results compared with that in neutral solutions. Antibacterial activities
are presented in Fig. 3. As can be seen from Fig. 3, the MIC against planktonic bacteria of the prepared NPs at various pH
of AgNPs-LA-OB at pH 4.7 against E. coli and S. aureus were values were further studied with MBC studies using a plate
1.09 mg mLꢀ1 and 0.55 mg mLꢀ1, respectively, (expressed as the counting method. Fig. 4 shows the bacterial growth on agar
total Ag concentration), which was much lower than that of plates with serial dilutions of AgNPs. The MBC shown is the
AgNPs-LA-OB at pH 7.4 (4.37 mg mLꢀ1 and 2.17 mg mLꢀ1
,
minimum silver concentration at which no visible bacterial
respectively). The MIC of the nanocomposites was also deter- colonies were present on the plates. The MIC and the MBC
mined using a microserial dilution method with tetrazolium red values are summarized in Table 2. It was concluded from Fig. 4
as indicator. Enhanced antibacterial activities were also found and Table 2 that the MBCs of AgNPs-LA-OB at pH 4.7, pH 5.5
for AgNPs-LA-OB at an acidic solutions (pH 4.7 or pH 5.5) and pH 7.4 were 1.09 ppm, 2.17 ppm and 4.34 ppm, respectively,
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for E. coli whereas those for S. aureus were 1.09 ppm, 1.09 ppm
and 4.34 ppm, respectively. The MBC and MIC results all
demonstrated enhanced antibacterial performance at lower
pH values for the pH induced surface switchable AgNPs. This
pH activated enhanced antibacterial behavior could be attributed
to the positive surface charge of the AgNPs-LA-OB in an acidic
solution which can induce stronger interaction between AgNPs
and microbes (negatively charged).
3.3 Visual investigation of the interaction of AgNPs-LA-OB
and microbes using TEM
In order to study the interaction between the NPs and bacteria,
bacteria cells incubated in the presence of AgNPs-LA-OB were
examined using a TEM. The morphologies of the microorganisms
before and after treatment with AgNPs-LA-OB were studied using
TEM and the results are shown in Fig. 5. The E. coli (A) remained
intact without AgNPs. However, the membranes of the bacteria
had an obviously wrinkled and twisted morphology which resulted
in destruction of microbes when interacted with AgNPs-LA-OB
(Fig. 5B and C). Importantly, some AgNPs were adhered onto the
membranes of the bacteria or penetrated into the bacteria cells,
which caused visible membrane destruction and the leakage
of intracellular substances. Based on these results, it was hypo-
thesized that under an acidic infection environment, AgNPs-LA-
OB exhibited a pH responsive transition from a negative charge
to a positive charge which can very strongly and readily adhere
on the negatively charged cell wall by means of an electrostatic
Fig. 4 Photographs of the bactericidal test in the presence of various
concentrations of AgNPs-LA-OB against S. aureus and E. coli at pH 7.4, pH
5.5 or pH 4.7.
Table 2 MIC and MBC values of AgNPs-LA-OB under different pH conditions
MIC (ppm)
MBC (ppm)
pH value
E. coli
S. aureus
E. coli
S. aureus
pH 7.4
pH 5.5
pH 4.7
4.34
2.17
1.09
2.17
1.09
0.55
4.34
2.17
1.09
4.34
1.09
1.09
Fig. 5 Comparative TEM images of microbes of E. coli (A–D) in the absence (A) and presence (B–D) of AgNPs-LA-OB for 8 h. The yellow circles highlight
AgNPs-LA-OB adhered to the microbial surface in (D). The rupture of the microbes’ structure is highlighted by the red circles in (B and C).
Fig. 6 Percentage of hemolysis measured using a spectrophotometer (A) and photographs of RBCs after exposure to AgNPs-LA-OB for 2 h (B).
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of the supernatants is shown in Fig. 6. The AgNPs-LA-OB
solution did not cause any observable hemoglobin release at
the concentration of 4.37 mg mLꢀ1 which is a lethal dose to kill
both Gram-negative and Gram-positive bacteria and is far lower
than those reported in the literature for bacteria of the same
size.59,60 The large, negatively charged surface of the small
AgNPs may account for the low hemolysis activity. Although
the AgNPs-LA-OB solution efficiently disrupts the microbial
walls/membranes under the infection site, it does not damage
the RBC membranes in healthy tissues which was attributed to
the pH responsive zwitterionic charge of the AgNPs. Therefore,
AgNPs-LA-OB showed satisfactory selectivity for bacterial cells
over RBCs.
3.5 Antibiofilm activities of AgNPs-LA-OB
AgNP–LA-OB dispersed into PBS with a pH of 7.4, 5.5 and 4.7
resulted in AgNP–LA-OB with different surface charges. A seven
day old S. aureus biofilm was treated with 200 mL of AgNP–LA-OB
solutions for 16 h. A standard plate counting assay, Live/Dead
staining assay and SEM observation were used to evaluate the
antibiofilm activities of AgNP–LA-OB in different acidic micro-
environments. The standard plate counting assay (Fig. 7) illustrated
that about 30% bacteria survive after 16 h treatment with AgNP–LA-
Fig. 7 Percentages of survival rates of S. aureus in biofilms for bacteria
colonies before and after treatment with AgNPs-LA-OB.
interaction, causing the destruction of the microbial membrane
and leading to cell lysis and irreversible apoptosis.
3.4 Hemolytic activity
The biocompatibility assay was also investigated to validate OB at pH 7.4 whereas less than 10% bacteria survive at treatment in
the increasing use of AgNPs in medical applications and the acidic solutions, indicating the outstanding bactericidal activities of
preliminary hemocompatibility of the prepared AgNPs-LA-OB AgNP–LA-OB under acidic environment as a result of a surface
was evaluated using a hemolysis assay. The hemoglobin released charge switch to positive in acidic solutions. The antibiofilm
from mice RBCs after incubation with different concentrations of activities of AgNP–LA-OB were further examined visually using
AgNPs-LA-OB solution was evaluated using UV-vis absorbance at a CLSM observation of cells stained with a LIVE/DEAD Biofilm
wavelength of 570 nm.
Viability kit. The live bacteria with intact cell membranes dis-
The concentration of AgNPs-LA-OB solution varies from played a green fluorescence and the dead bacteria with damaged
0.55 mg mLꢀ1 to 17.37 mg mLꢀ1 after incubation with RBCs for cell membranes showed red fluorescence in the CLSM images. As
2 h. The photograph of RBCs and spectrophotometric analysis shown in Fig. 8, untreated controls were characterized by intact
Fig. 8 The confocal microscopy images of S. aureus biofilms before treatment with AgNPs-LA-OB (A) and treatment with AgNPs-LA-OB at pH 7.4 (B),
pH 5.5 (C) and pH 4.7 (D) after 16 h incubation were stained using LIVE/DEAD BacLight Bacterial Viability kits. Live (green) and dead (red) bacteria were
imaged using CLSM. Images A, B, C and D show the top view and images A1, A2, B1, B2, C1, C2, D1, D2 are three-dimensional reconstructions of z-stacks
collected across the biofilm thickness. Scale bars: 50 mm.
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deep penetration of the particles within the bacterial biofilm.
Therefore, the AgNPs-LA-OB can be potentially used as new anti-
bacterial materials to combat bacterial infections of their biofilms.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the Applied Basic Research
Programs Foundation of Sichuan Province (No. 2015JY0126) and
by the Functional Polymer Innovation Team Project, Southwest
Minzu University (No. 14CXTD04).
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