Facile synthesis of a biocompatible silver nanoparticle derived tripeptide supramolecular hydrogel for antibacterial wound dressings

Article abstract of DOI:10.1039/c5nj01981h

Realizing the widespread demand for biocompatible supramolecular hydrogel based wound dressings, we have developed an N-terminally 2(naphthalen-6-yl)acetic acid (Nap) protected Phe-Phe-Cys peptide (Nap-FFC) using a liquid phase method. This Nap-FFC peptid

Full text of DOI:10.1039/c5nj01981h

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Facile synthesis of a biocompatible silver
nanoparticle derived tripeptide supramolecular
Cite this:DOI: 10.1039/c5nj01981h
hydrogel for antibacterial wound dressings†
Turibius Simon, Chung-Shu Wu, Jie-Chuan Liang, Chieh Cheng and Fu-Hsiang Ko*
Realizing the widespread demand for biocompatible supramolecular hydrogel based wound dressings,
we have developed an N-terminally 2(naphthalen-6-yl)acetic acid (Nap) protected Phe–Phe–Cys peptide
(Nap-FFC) using a liquid phase method. This Nap-FFC peptide was used to design a supramolecular
hydrogel via a self-assembling process. The Nap-FFC short peptides produced stable and transparent
silver nanoparticle-based hydrogels (AgNPs@Nap-FFC) wherein the self-assembled Nap-FFC nanofibers
acted as scaffolds for the mineralization of silver nanoparticles (AgNPs) and stabilizers of the synthesized
AgNPs. The resultant AgNPs@Nap-FFC nanocomposites were characterized using ultraviolet-visible
spectrophotometry (UV-vis spectroscopy), Fourier transform infrared spectroscopy (FT-IR), transmission
electron microscopy (TEM) and scanning electron microscopy (SEM) combined with energy-dispersive
X-ray spectroscopy (EDX) studies. The AgNPs@Nap-FFC nanocomposites showed excellent monodispersity,
long term stability, and functional flexibility in comparison to other AgNP based nanocomposites.
Furthermore, AgNPs@Nap-FFC exhibited strong inhibition against both Gram-positive (methicillin-resistant
Staphylococcus aureus) and Gram-negative (Acinetobacter baumannii) bacteria and, most importantly, they
showed favorable biocompatibility towards human cervical carcinoma cells (HeLa cells). Hence, this study
implies that AgNPs@Nap-FFC nanocomposites can easily be prepared in a cost-effective manner and can
be used effectively for future antibacterial wound dressings.
Received (in Montpellier, France)
30th July 2015,
Accepted 8th December 2015
DOI: 10.1039/c5nj01981h
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in aqueous medium, due to the release of surface bound Ag+
ions thereby, causing the loss of antibacterial activity and
Introduction
Much attention has been devoted to the pharmacology of silver inducing cellular toxicity and reactive oxygen species (ROS)
nanoparticles (AgNPs) in terms of their large surface area to production to normal cells upon overtime exposure.8,9 Hence
volume ratio, physicochemical properties, antioxidant activity there is a need to synthesize stable biocompatible AgNPs in
and low toxicity to mammalian cells.1–3 AgNPs induce the order to reduce their cellular toxicity in health care or biomedical
production of hydroxyl radicals (ꢀOH) to kill bacterial cells, applications.10,11 Different polymer nanocomposites such as poly-
without interacting with the multidrug-resistant pumps in (oxyethylene)-segmented imide (POEM), poly(styrene-co-maleic
bacteria that produce toxins to inhibit antibiotics like ampicillin. anhydride)-grafting poly(oxyalkylene) (SMA) and poly(vinyl alcohol)
This suggests that resistance to AgNPs is less likely to occur in (PVA) have been employed to stabilize AgNPs and been used for
bacteria and hence it can be used as a new generation antimicrobial biomedical applications.12 But the stabilization of polymer based
agent to overcome the drug resistance seen with Gram-negative nanocomposite materials requires prolonged synthesis steps and
and Gram-positive bacteria.4,5 This unique character enabled high cost, and shows inadequate biocompatibility.13
AgNPs to be used in a wide variety of products such as refrigerators,
To improve the biocompatibility and to shorten the synthesis
clothes, toothbrushes, and cosmetics.6 AgNPs have been shown to steps, proteins or artificial self-assembling peptide-based scaf-
have superior cytotoxicity against a wide range of microorganisms folds are employed to synthesise peptide based nanocomposite
when compared to many metal nanoparticles.7 However, bare materials through a biomineralization mechanism.14 Common
AgNPs (e.g. sodium borohydride/citrate reduced AgNPs) are unstable biomimetic peptides include the fibronectin-derived RGDS
sequence for therapeutic cell delivery,15 the laminin derived
IKVAV sequence16 used for bone regeneration and wound healing,
etc. In addition, self-assembled peptide-based hydrogels could be
Department of Materials Science and Engineering, National Chiao Tung University,
1001 University Road, Hsinchu, Taiwan 300, Republic of China.
used for various therapeutic approaches such as the regeneration
E-mail: fhko@mail.nctu.edu.tw; Tel: +886-35712121 ext 55803
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nj01981h of enamel, cartilage, and the central nervous system, as well as
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delivery vehicles, transplantation of islets, wound-healing and Synthesis of a biocompatible peptide
cardiovascular therapies.17,18 Among the self-assembling peptides
N-terminally Nap protected peptide Nap-FFC (Nap-Phe–Phe–Cys)
was synthesized using a liquid phase method.21
used,19 the diphenylalanine (FF) peptide and its derivatives can be
more easily self-assembled into various nanostructures than other
lengthy peptides applied for biomedical applications.20 Proceeding
from these research reports, we decided to introduce an efficient
peptide-tunable hydrogel, co-fabricated with AgNPs. Accordingly,
an N-terminally protected FF peptide derivative (Nap-FFC) was
synthesized in laboratory-scale, which showed a robust metal
binding ability with AgNPs and was able to form stable and
transparent AgNPs@Nap-FFC hydrogels. The synthetic reactions
were mild and environment friendly. Furthermore, bioassays
revealed that the synthesized AgNPs@Nap-FFC nanocomposites
possess an effective and long term antibacterial activity against
both Gram-positive bacteria and Gram-negative bacteria. Most
importantly, the AgNPs@Nap-FFC hydrogels were proven to be
non-toxic to human cells, and therefore this approach would be
a versatile platform in mechanobiology. Due to the strong
bacterial inhibition activity, this novel AgNPs@Nap-FFC hydrogels
could be an attractive antibacterial material for use as wound
dressings in biomedical applications.
Synthesis of Nap-F
2(Naphthalen-6-yl)acetic acid (Nap, 372 mg, 2 mmol) and
N-hydroxysuccinimide (NHS, 230 mg, 2 mmol) were dissolved
in 20 mL chloroform and N,N0-dicylcohexylcarbodiimide (DCC,
432 mg, 2.1 mmol) was added. The mixture was stirred at room
temperature for 5 h, then the resulting solid was filtered off,
and the filtrate was subjected to rotary evaporation. The crude
product obtained (Nap-NHS) was used without purification.
L-Phenylalanine (330 mg, 2 mmol) and sodium carbonate
(Na2CO3, 424 mg, 4 mmol) were dissolved in 8 mL of water,
the solution of the crude product (Nap-NHS) (dissolved in
20 mL acetone) was added, and the resulting reaction mixture
was stirred at room temperature overnight. The reaction mixture
was subjected to rotary evaporation, and then 20 mL of water was
added. The filtrate was acidified to pH 3 and the resulting
product obtained by filtration was further purified using a flash
column with chloroform–methanol as the eluent. Compound
Nap-F (white powder) was collected with 58% yield. 1H NMR
(300 MHz, DMSO-d6) d (ppm): 8.44 (d, J = 8.1 Hz, 1NH), 7.86–7.75
(m, 3H), 7.64 (s, 1H), 7.46 (t, J = 6 Hz, 2H), 7.27–7.18 (m, 6H), 4.42
(t, J = 4.2 Hz, 1H), 3.57 (d, 2H), 3.09–2.82 (m, 2H). HRMS (ESIꢁ):
calculated for C21H19NO3 333.1365 found [M ꢁ H]ꢁ 332.1281.
Materials and methods
Chemicals and reagents
2(Naphthalen-6-yl)acetic acid (Nap), N,N0-dicylcohexylcarbo-
diimide (DCC), N-hydroxysuccinimide (NHS), L-phenylalanine
(Phe), L-cysteine (Cys) and sodium carbonate (Na2CO3) were
purchased from Alfa Aesar and used without further purification.
Silver nitrate (AgNO3), sodium borohydride (NaBH4), chloroform
(CHCl3) and methanol (CH3OH) were obtained from Aldrich,
U.S.A. Metal salts such as ZnCl2, MgCl2, AgCl2, FeCl2, CoCl2, CdCl2,
BaCl2, LiCl, NiCl2, KCl and CaCl2 were from Baker Analyzed ACS
Reagent, U.S.A. Bacterial strains (methicillin-resistant Staphylococcus
aureus and Acinetobacter baumannii) and HeLa cell lines were
from the BCRC (Bioresource Collection and Research Center)
Taiwan. Bacterial culture media such as trypticase soy broth
(TSB) and trypticase soy agar (TSA) were purchased from Becton,
Dickinson and Company. The medium used for the culture of
HeLa cells was Dulbecco’s modified eagle medium (DMEM-
Thermo Scientific).
Synthesis of Nap-FF
Compound Nap-F (333 mg, 1 mmol) and NHS (115 mg, 1 mmol)
were dissolved in chloroform (10 mL) and DCC (216 mg,
1.05 mmol) was added. The mixture was stirred at room
temperature for 5 h, then the resulting solid was filtered off,
and the filtrate was subjected to rotary evaporation. The crude
product obtained (Nap-F-NHS) was used without purification.
L-Phenylalanine (165 mg, 1 mmol) and Na2CO3 (212 mg, 2 mmol)
were dissolved in 4 mL of water, the solution of crude product
(Nap-F-NHS) was dissolved in 10 mL of acetone, and then the
resulting reaction mixture was stirred at room temperature
overnight. The reaction mixture was subjected to rotary evaporation,
and then 10 mL of water was added. The filtrate was acidified to
pH 3, and the resulting product obtained by filtration was
further purified using a flash column with chloroform–methanol
as the eluent. The dipeptide Nap-FF (white powder) was collected
with 50% yield. 1H NMR (300 MHz, DMSO-d6) d (ppm): 8.29–8.49
(m, 3H), 7.42–7.86 (m, 3H), 7.10–7.28 (m, 8H), 4.44–4.59 (m, 2H,
–CH), 2.95–3.10 (m, 3H), 2.68–2.92 (m, 3H). HRMS (ESIꢁ):
calculated for C30H28N2O4 480.2049 found [M ꢁ H]ꢁ 479.1966.
Instruments and product characterization
The molecular structures of Nap-F, Nap-FF, and Nap-FFC were
elucidated via nuclear magnetic resonance spectroscopy (NMR,
Bruker 300 MHz spectrometer) and high resolution mass analysis
(HRMS, Waters Premier XE instrument with ESI source). The ionic
interactions of Nap-FFC with various metal ions were determined
using Fourier transform infrared spectroscopy (FTIR, Perkin Elmer
Synthesis of Nap-FFC
spectrometer 100 FT-IR SPECTRUM ONE) and compared deduced Compound Nap-FF (240 mg, 0.5 mmol) and NHS (57.5 mg,
reactions. The silver nanoparticles (AgNPs) and silver based Nap- 0.5 mmol) were dissolved in chloroform (6 mL) and DCC
FFC nanocomposites (AgNPs@Nap-FFC) were characterized via (107 mg, 0.52 mmol) was added. The mixture was stirred at
UV-vis spectroscopy (HITACHI, U-3310), scanning electron micro- room temperature for 5 h, then the resulting solid was filtered
scopy (SEM, JEOL, JSM-6700) and transmission electron micro- off, and the filtrate was subjected to rotary evaporation. The
scopy (TEM, JEOL, JEM-2100).
crude product obtained (Nap-FF-NHS) was used without purification.
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L-Cysteine (60.5 mg, 0.5 mmol) and Na2CO3 (106 mg, 1 mmol) at room temperature and the bacterial growth rates were
were dissolved in 2 mL of water; the solution of crude product determined by measuring the OD at 600 nm.
(Nap-FF-NHS) dissolved in 6 mL of acetone was added. And the
Biocompatibility studies using an MTT assay
resulting reaction mixture was stirred at room temperature
overnight. The reaction mixture was subjected to rotary evaporation,
and then 10 mL of water was added. The filtrate was acidified to pH
3 and the resulting product obtained by filtration was further
purified using a flash column with chloroform–methanol as the
eluent. The final compound Nap-FFC (white powder) was collected
with 30% yield. The 1H NMR (300 MHz, DMSO-d6) d (ppm):
12.93 [s (br), 1H (–COOH)], 8.25–8.48 [m, 6H (–NH & aromatic)]
7.30–7.48 [m, 3H, (aromatic)], 7.10–7.24 [m, 8H (aromatic)],
4.44–4.59 [m, 3H (–CH)], 2.95–3.09 [m, 4H (–CH2)], 2.50–2.84
[m, 4H (–CH2)], 1.761 [s (br), 1H (SH)]. HRMS (ESIꢁ): calculated
for C33H33N3O5S 583.2141 found [M ꢁ H]ꢁ 582.2070.
In order to evaluate the cytotoxic activity of AgNPs@Nap-FFC
nanocomposites, human cervical carcinoma (HeLa) cells were
used in this study. HeLa cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum (FBS) and 1ꢂ antibiotics solution, and cultured
at 37 1C in a humidified incubator containing 5% CO2. For the
cytotoxicity assay, cells were grown in 96-well plates (flat bottom) at
a density of 1.5 ꢂ 104 cells per well with 100 mL of the medium.
After overnight culturing, the culture medium was aspirated and
fresh media containing the AgNPs@Nap-FFC nanocomposites at
concentrations ranging from 10 to 100 mg mLꢁ1 were added and
incubated for 24 h. In order to determine the viability of the HeLa
cells, 100 mL of the MTT solution (5 mg mLꢁ1) diluted in serum
free DMEM medium was added, and incubated at 37 1C for 4 h.
After 4 h of incubation, purple colored crystals formed and were
dissolved in DMSO and the absorbance was measured at 570 nm
using a Multiskan GO micoplate reader. All experiments were
independently performed at least three times. The results were
presented as percentage of viable cells against the increasing
dose of the AgNPs@Nap-FFC nanocomposites. The untreated
cells served as control.
Gelation properties of Nap-FFC
Different concentrations of Nap-FFC (w/v) in aqueous solution
under different pH conditions were evaluated for their gelation
properties.
Synthesis of silver nanoparticles (AgNPs)
62.5 mL of 0.1 M silver nitrate (AgNO3) and freshly prepared ice
cold 300 mL of 5 mM sodium borohydride (NaBH4) were added
in 25 mL of distilled water with continuous stirring. The reaction
was carried out at room temperature for 30 minutes. The reaction
mixture slowly turned to a yellow coloured solution, indicating the
formation of AgNPs.22 The prepared AgNP solution was stored at
4 1C for further study.
Results and discussion
Physicochemical characterization of the Nap-FFC peptide
Synthesis of AgNPs@Nap-FFC nanocomposites
The preparation method of the Nap protected FFC peptide
62.5 mL of 0.1 M silver nitrate (AgNO3) was added to 25 mL of a (Nap-Phe–Phe–Cys) via liquid phase approach is illustrated in
120 mM Nap-FFC solution (pH 12). The mixture was gently Fig. S1 (ESI†). The primary molecular structure of Nap-F, Nap-FF,
stirred for 30 minutes at room temperature. Then, freshly and Nap-FFC were confirmed using the NMR spectra of each
prepared cold 300 mL of 5 mM sodium borohydride (NaBH4) compound (Fig. S2–S4, ESI†). The molecular structures of the
was added to the reaction mixture. The reaction mixture then slowly synthesized peptides, Nap-F, Nap-FF, and Nap-FFC, were further
turned to a brown colour solution, indicating the formation of silver verified via high resolution mass analysis (Fig. S5–S7, ESI†). The
nanoparticle based Nap-FFC nanocomposites (AgNPs@Nap-FFC).
data obtained from the NMR and high resolution mass spectra
revealed the exact peptide sequence identity peak and mass value
of those compounds. The supramolecular Nap-FFC peptide
Antibacterial activity determination
The antibacterial activity of the AgNPs@Nap-FFC hydrogel was structure showed two different moieties: hydrophobic and hydro-
evaluated via direct contact with Gram positive and Gram philic. The hydrophobic group promotes the self-assembly process
negative bacteria. Common drug resistant bacterial strains such to form hydrogels. On the other hand, the hydrophilic moiety
as methicillin-resistant Staphylococcus aureus and Acinetobacter exhibits many carboxylic acid and thiol groups on the supra-
baumannii were tested. In brief, bacterial strains cultured in molecule nanofiber (Nap-FFC) surface, which can act as silver
2 mL of trypticase soy broth (TSB) were treated with the AgNPs@ metal ion binding sites and nucleation sites for the growth of
Nap-FFC hydrogel at concentrations of 40 and 80 mg mLꢁ1 of silver nanoparticles. The synthesized Nap-FFC peptide has free
bacterial medium. Bacterial growth rates were determined by carboxylic acid and thiol groups which enable the tuning of the
measuring the optical density (OD) at 600 nm using a spectro- peptide nature based on the desired conditions. These peptides
photometer (cell density meter). For the bactericidal activity can self-assemble to form supramolecular hydrogels with respect
assay, the bacteria were grown on TSA plates at 37 1C for 24 hours to different conditions, such as the peptide weight percentage
to obtain the late-log growth phase (OD600 = 2.0). The plates concentration (w/v), pH conditions and mineralisation with metal
were incubated with a 1% (w/v) Nap-FFC hydrogel pad and ions. For this study, we have taken the Nap moiety as a protecting
AgNPs@Nap-FFC nanocomposites (80 mg mLꢁ1). Meanwhile, group since it is present in clinically approved drug molecules
the 1% Nap-FFC (w/v) hydrogel pad incubated under the same such as propranolol and naphazoline, and are more biocompatible
environments was used as a control. The agar plates were incubated than the Fmoc or pyrene groups.23 The hydrophilic moiety of
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Fig. 1 Schematic illustration for the preparation process of the AgNPs@Nap-FFC nanocomposites.
Nap-FFC has numerous carboxylic acid and thiol groups on completely soluble at pH 4 10, due to the stable deprotonation
the peptide nanofiber surface, which act as nucleation sites for of the compound under highly alkaline conditions. Interestingly,
the growth of nanoparticles and as metal binding sites. The the Nap-FFC hydrogel (1% w/v) was semi-transparent at pH
mineralization process of silver ions using the peptide nanofibers ranging from 7 to 10, but in the acidic pH range (5 to 6) the
are shown in Fig. 1. We proposed the synthetic method of hydrogel was opaque. In addition, at low pH (pH = 4) the 1%
supramolecular AgNP entrapped hydrogel (AgNPs@Nap-FFC) (w/v) Nap-FFC hydrogel was starting to form a precipitate, which
nanocomposites. The above mechanism displays more advantages might be because of stable protonation. The formation of a
than other methods due to the ease of fabrication, good bio- hydrogel within a narrow pH range (5 to 6) likely arises from a
compatibility, cost effectiveness, the acceleration of the wound balance of protonation/deprotonation of the C-terminal carboxyl
healing process, molecular recognition capability and functional group.25–27
flexibility.2,24 Metal ions can coordinate with negatively charged
carboxylic acid groups and thiol groups through an electrostatic
interaction process and are reduced to metal atoms. The Ag ions
were able to coordinate more effectively with Nap-FFC and
henceforth we used sodium borohydride which subsequently
initiated the generation of Ag nuclei that finally grow into
AgNPs along with peptide nanofibers. This mechanism of bio-
mineralisation enabled the preparation of the silver nanoparti-
cle based Nap-FFC nanocomposites (AgNPs@Nap-FFC).
FT-IR analysis
The interaction of the Nap-FFC peptide with silver ions was
studied using FT-IR analysis, as shown in Fig. S8 (ESI†). The IR
peak indicates the presence of CQO groups at 1670 cmꢁ1 and
the S–H stretching of Nap-FFC at 2569 cm .
ꢁ1 28 Interestingly, the
S–H and CQO stretching bands at 2569, 1670 cmꢁ1 of Nap-FFC
were absent due to the binding of silver ions to the carbonyl and
thiol terminals of Nap-FFC. According to the above results, the
Nap-FFC peptide displayed a carboxylic acid and thiol group on
the supramolecule nanofiber surface, which offers the strong
interaction with silver ions.29,30
Gelation property of Nap-FFC
Different pH induced phase transitions of 1% Nap-FFC hydrogel
(w/v) at 30 1C are shown in Fig. 2. The 1% w/v Nap-FFC was
Stability of the AgNPs@Nap-FFC nanocomposite
To examine stability, the Nap-FFC peptide nanocomposites
(AgNPs@Nap-FFC) produced via the in situ reduction method
was compared with bare AgNPs (NaBH4 reduced) using UV-vis
absorption spectroscopy. The UV-vis absorption spectra of the
bare AgNPs and AgNPs@Nap-FFC show a surface plasmon
resonance (SPR) absorption peak at 400 nm and 420 nm,
respectively, as shown in Fig. 3(A) and (B). The characteristic
SPR peaks of the bare AgNPs at 400 nm showed large decrements
in the absorption intensity within seven days due to the serious
aggregation of AgNPs that usually leads to a significant decrease in
the antibacterial effect. The SPR band of the AgNPs@Nap-FFC
nanocomposites is somewhat longer than that of typical bare
AgNPs and this red-shift in wavelength is due to the high
dielectric constant of the Nap-FFC peptide, which lowers the
plasmon frequency.31,32 On the other hand, the UV-vis spectra of
the AgNPs@Nap-FFC nanocomposites showed only a slight change
in the shifting of the SPR band (B2 nm) over a time period of
30 days. Thus, the AgNPs@Nap-FFC peptide nanocomposites
Fig. 2 Optical images of the Nap-FFC hydrogel ([Nap-FFC] = 1 wt%). The
Nap-FFC hydrogel (1 wt%) exists as (A) a precipitates at pH o 4.0, (B and C)
a strongly opaque hydrogel at a pH range of about 4.0 to 6.0, (D–G) a
weak transparent hydrogel at a pH range of about 7.0 to 10.0, and (H) a
clear solution at pH 4 10.
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Fig. 3 The absorption spectra and SEM analysis of the AgNPs (A and C) and AgNPs@Nap-FFC nanocomposite (B and D) at pH 7.4. Scale bar: 100 nm.
specify long term high stability. Furthermore, the morphologies
of the bare AgNPs and AgNPs@Nap-FFC peptide nanocomposites
were examined via SEM analysis and the images are shown in
Fig. 3(C) and (D), respectively. The average size of the bare AgNPs
and mineralised AgNPs obtained from the Nap-FFC nanofibers are
approximately B15 nm and B10 nm, respectively. TEM analysis
further confirms the size of the AgNPs and AgNPs@Nap-FFC
peptide nanocomposites (shown in Fig. S9, ESI†). Therefore, the
newly developed AgNPs@Nap-FFC peptide nanocomposite has
a high monodispersity and long term stability even in aqueous
medium.
For antibacterial applications, the hydrogel was formed by
combining 80 mg mLꢁ1 AgNPs@Nap-FFC peptide nanocomposites
(liquid form) with 1% Nap-FFC (no AgNPs present) at pH 12. Also,
the effect of gelation of AgNPs@Nap-FFC was systematically
studied at different periods (shown in Fig. S10, ESI†). According
to the photographic images, the time dependent gelation can
be seen when 80 mg mLꢁ1 AgNPs@Nap-FFC was added to 1%
Fig. 4 The SEM analysis (A and B) of 1% (w/v) Nap-FFC without/with
Nap-FFC, which was able to form hydrogels overnight. No
characteristic gelation was seen at 30 min even in the presence
of AgNPs@Nap-FFC but hydrogel formation was observed after
overnight incubation, due to the self-assembly process of
Nap-FFC, with respect to time. On the contrary, we also observe
that the clear solution of 1% Nap-FFC (w/v) without AgNPs@
Nap-FFC does not change phase after an overnight time period.
The SEM image (Fig. 4A and B), ensured the morphology of
the 1% (w/v) Nap-FFC with/without AgNPs@Nap-FFC nano-
80 mg mLꢁ1 AgNPs@Nap-FFC. The EDX analysis (C) of 1% (w/v) Nap-FFC
containing 80 mg mLꢁ1 AgNPs@Nap-FFC. Scale bar: 100 nm.
peak at 0.2 and 0.4 keV, respectively, which is related to the
amino acid of Nap-FFC.
Bacterial kinetic test
composites (80 mg mLꢁ1) and we noticed the uniform dispersion A bacterial kinetic test was performed to determine the anti-
of AgNPs on the surface of the Nap-FFC nanofiber. Further bacterial activity of the Nap-FFC and AgNPs@Nap-FFC nano-
confirmation of the presence of AgNPs in NapFFC nanocomposites composites. As shown in Fig. 5, a comparison of the growth
was done via EDX analysis (Fig. 4C). The peak in the EDX curves of the control bacteria to the test bacteria shows that
spectra indicates the presence of silver at 3 keV and the C and O Nap-FFC at 120 mM did not alter the growths of S. aureus and
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Fig. 5 Bacterial growth curves in Luria–Bertani (LB) media with Nap-FFC or AgNPs@Nap-FFC nanocomposites stored at 4 1C for 7 days. Comparison of
the antibacterial activity of Nap-FFC and AgNPs@Nap-FFC against methicillin-resistant Staphylococcus aureus (A and B) and Acinetobacter baumannii
(C and D).
A. baumannii, suggesting that it has no inhibitory effect
on these bacteria (Fig. 5A and C). On the other hand, even
40 mg mLꢁ1 of AgNPs@Nap-FFC was able to slow down the
growth of both S. aureus and A. baumannii at 12 and 28 h,
respectively. Moreover, when the concentration of AgNPs@Nap-
FFC was increased to 80 mg mLꢁ1 (Fig. 5B and D), the growth of
both bacteria showed strong long-term inhibition, revealing
that these nanocomposites can be an attractive biomaterial for
applications such as wound dressing.
Bacterial spread plate method
The antibacterial activity of the Nap-FFC and AgNPs@Nap-FFC
nanocomposites were further ensured via the bacterial spread
plate method. For this, Nap-FFC without/with AgNPs@Nap-FFC
was layered over a lawn of S. aureus and A. baumannii in a TSA
plate, and incubated at room temperature for 12 and 24 hours.
After 12 hours of incubation, only AgNPs@Nap-FFC (80 mg mLꢁ1
)
produced a clearly visible area of inhibition (Fig. 6 and 7),
suggesting an excellent bacterial inhibition property due to the
release of a large amount of nanosilver particles (Fig. 6 and 7).
The antibacterial activity was maximal and then decreased
after 24 hours of incubation due to a gradual decrease in
the amount of nanosilver particles released, which was also
reported in previous studies.33 Henceforth, the resumed growth
of both bacteria occurred when the concentration of nanosilver
decreased below the minimum inhibitory concentration. So
Fig. 6 Schematic illustration of the antibacterial test of the Nap-FFC and
AgNPs@Nap-FFC hydrogels; Petri plates with Luria–Bertani (LB)-agar
inoculated with methicillin-resistant Staphylococcus aureus, showing variable
numbers of colonies when supplemented with 1% (w/v) Nap-FFC with/
without 80 mg mLꢁ1 AgNPs@Nap-FFC.
Biocompatibility of the AgNPs@Nap-FFC nanocomposites
we speculate that under different time phases, the decreasing To further test the potential applications of the AgNPs@Nap-
nanosilver concentration may result in lower antibacterial FFC nanocomposites, we evaluated the toxic effects of these
activity.
nanocomposites in human HeLa cells using an MTT assay.
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hydrogel can provide the moisture environment to enhance
human cell growth. Overall, 80–90% of the cells were found
to be viable at 40 and 80 mg mLꢁ1 concentrations of the
AgNPs@Nap-FFC nanocomposites. These data suggested that the
AgNPs@Nap-FFC nanocomposite has a favourable biocompatibility
towards human cells.
Conclusions
In summary, we report the synthesis of Nap-FFC peptide based
nanofibers which were utilized to produce AgNPs@Nap-FFC
nanocomposites via a mineralization process. After the successful
synthesis, Nap-FFC and AgNPs@Nap-FFC were characterized via
NMR, high resolution mass analysis, SEM, TEM, FTIR and EDX.
This silver based nanocomposite (AgNPs@Nap-FFC) offered an
extended stability of approximately 30 days compared to that of
bare AgNPs (NaBH4 reduced). Also, the factors which influence the
formation of hydrogels such as the effect of gelation at different
pH values and mineralization of the Nap-FFC peptide with various
metal ions were systematically studied. The results revealed that
the interaction of the Nap-FFC peptides with Ag+ ions was more
pronounced due to the presence of carboxyl and thiol groups in
Nap-FFC, resulting in the formation of AgNPs@Nap-FFC. The
optimum conditions to prepare AgNPs@Nap-FFC are 1% Nap-FFC
(w/v) at pH 12 at 30 1C. For the antibacterial study, a hydrogel pad
was prepared by mixing AgNPs@Nap-FFC (80 mg mLꢁ1) in 1%
Nap-FFC (w/v) peptide. These nanocomposite pads showed a strong
antibacterial effect against both Gram-positive and Gram-negative
bacteria. Moreover, the nanocomposites displayed significant
biocompatibility towards HeLa cells. Therefore, the prepared
AgNPs@Nap-FFC peptide nanocomposites are promising candidates
for antibacterial soft materials for future tissue engineering and
wound dressing.
Fig. 7 Schematic illustration of the antibacterial test of Nap-FFC and
AgNPs@Nap-FFC hydrogels; Petri plates with Luria–Bertani (LB)-agar
inoculated with Acinetobacter baumannii, showing variable numbers
of colonies when supplemented with 1% (w/v) Nap-FFC with/without
80 mg mLꢁ1 AgNPs@Nap-FFC.
After 24 hours of incubation, we found that the AgNPs@Nap-FFC
nanocomposites showed no or little effect on HeLa cells at
concentrations up to 80 mg mLꢁ1 (Fig. 8). Even after increasing
the concentration of the AgNPs@Nap-FFC nanocomposite up to
100 mg mLꢁ1, they displayed only a mild decrease in human cell
viability ensuring its lower cytotoxicity and proving to reduce the
risk of harmful side effects. Moreover, the Nap moiety adopted
to prepare this peptide has been used in clinically approved
drug molecules such as propranolol and naphazoline,23 which
adds more safety to this nanocomposites. The supramolecular
Acknowledgements
The authors are grateful to the National Taiwan University
Hospital Hsinchu Branch and the Ministry of Science and
Technology of Taiwan for financially supporting this research
under the contract MOST 101-2113-M-009-007-MY3.
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