Materials Science and Engineering C 39 (2014) 177–185

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Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Eco-designed biohybrids based on liposomes, mint–nanosilver and

carbon nanotubes for antioxidant and antimicrobial coating
Marcela Elisabeta Barbinta-Patrascu a, Camelia Ungureanu b,⁎, Stefan Marian Iordache c, Ana Maria Iordache c,
Ioana-Raluca Bunghez d, Marius Ghiurea d, Nicoleta Badea b, Radu-Claudiu Fierascu d, Ioan Stamatin c
a
University of Bucharest, Faculty of Physics, Department of Electricity and Magnetism, Solid-State Physics, and Biophysics, 405 Atomistilor Street, P.O. Box MG-11,
Bucharest-Magurele 077125, Romania
b
University “Politehnica” of Bucharest, Faculty of Applied Chemistry and Materials Science, 1-7 Polizu Str., 011061 Bucharest, Romania
c
University of Bucharest, Faculty of Physics, 3Nano-SAE Research Centre, P.O. Box MG-38, Bucharest-Magurele 077125, Romania
d
National Institute for Research & Development in Chemistry and Petrochemistry (INCDCP-ICECHIM), 202 Spl. Independentei, Bucharest 060021, Romania

a r t i c l e i n f o a b s t r a c t

Article history: Noncovalent entities (consisting of liposomes, phyto-nanosilver and carbon nanotubes) with interesting
Received 7 November 2013 properties were constructed by a “green” bottom-up method. Phytosynthesis of silver nanoparticles using
Received in revised form 2 February 2014 the Mentha piperita extract combines the benefits of this herb with the interesting properties of silver.
Accepted 17 February 2014
The obtained silver-based biohybrids showed antioxidant and antimicrobial properties that have been con-
Available online 5 March 2014
siderable improved in the presence of carbon nanotubes. Thus the eco-designed bioconstructs consisting of
Keywords:
cholesterol-containing liposomes, phytonanosilver and carbon nanotubes exhibited high antioxidant activity
Biohybrids (AA = 90.8%) and have been shown to be strong biocides offering inhibition zone of 25 mm against
Biomimetic membranes Escherichia coli and 23 mm against Staphylococcus aureus and Enterococcus faecalis.
Silver nanoparticles © 2014 Elsevier B.V. All rights reserved.
Carbon nanotubes
Mentha piperita

1. Introduction Applying of green chemistry principles in silver nanoparticles

Nanotechnology has known a considerable progress in recent
years and opened new possibilities and study directions in many
fields: medicine (drug delivery systems, targeted therapy), biotechnol-
ogy (food safety, biosynthesis of metal nanoparticles by microorgan-
isms) and optoelectronics (biosensors). Many interesting properties
acquire a system whose size decreases from macro (bulk material) to
nano level. Synthesis and applications of noble metal nanoparticles are
parts of a vibrant and growing branch of nanotechnology [1].
Silver nanoparticles (AgNPs) are known to possess antimicrobial
properties, being able to inhibit the activity of viruses and bacteria
[2–4]. AgNPs have a broad range of applications including: medicine,
pharmacology, microelectronics, optics, disinfection, and water
treatment [1,5].
⁎ Corresponding author at: General Chemistry Department, Faculty of Applied
Chemistry and Materials Science, University Politehnica of Bucharest, 1-7 Polizu, 011061
Bucharest, Romania. Tel.: +40 723239120; fax: +40 318157561.
E-mail addresses: c_ungureanu@chim.pub.ro, ungureanucamelia@gmail.com
(C. Ungureanu).
(AgNPs) preparation has attracted significant interest in the last
years, offering the advantage of using natural products. Thus, herbal
extracts are attracting great interest due their antioxidant and anti-
microbial properties [6]. Thus, the plant extract use in AgNP synthesis
has opened new perspectives in nanotechnology, enabling the exploita-
tion of the vast phytotherapeutic potential. Phytofabrication of metallic
nanoparticles explores the synthesis of commercial nanoparticles using
‘green’ methods of synthesis based on natural materials: plant extracts
[7].
Among the hybrid organic–inorganic systems, the nanocomposites
containing noble metal nanoparticles [8] are attracting a growing
interest.
Carbon nanotubes (CNTs) are often used to obtaining composites
materials with novel features. As it is known, CNTs are carbon allo-
tropes with an interesting nanostructure based on honey comb type
graphene sheets (consisting of C\C sp2 bond, one of the strongest
bonds of nature) rolled-up in cylindrical nanotubes, are considered
the building blocks of new advanced biomaterials. Depending on num-
ber of graphene layers, three main classes of CNTs are known: single-
walled carbon nanotubes (SWCNTs), double walled carbon nanotubes
(DWCNTs) and multiwalled carbon nanotubes (MWCNTs). Since their
discovery, CNTs have attracted the attention of the scientific world

http://dx.doi.org/10.1016/j.msec.2014.02.038
0928-4931/© 2014 Elsevier B.V. All rights reserved.
178 M.E. Barbinta-Patrascu et al. / Materials Science and Engineering C 39 (2014) 177–185
due to their unusual properties that exceed those of any existing mate-
rials [9]. CNTs possess high mechanical strength, thermal and electronic
conductivity, and large active surface area, properties which are depen-
dent on their diameter and structure [10]. Research studies showed that
SWCNT is a perfect support material for nanoparticles and biological
material, with applications in sensors and waste water treatments [11].
The nanotubes are held together in bundles by van der Waals
forces, but the improvement properties of carbon nanotubes could
be achieved when CNTs are dispersed (functionalized) in liquid
media. Among the two ways of CNT functionalization: covalent and
noncovalent, the second is preferred because it preserves the sp 2
carbon network and allows the adsorption of different biomolecules
or metallic nanoparticles onto the nanotube surface via noncovalent
interactions, getting 3-D architectures with interesting properties.
This work presents a green bottom-up strategy to design novel
antimicrobial and antioxidant materials. The fabrication of coatings
with biocidal properties is a real exciting interest topic in nanotech-
nology [11]. This paper is a continuation of the research of prepara-
tion and characterization of biohybrids based on herbal silver
nanoparticles [12] using new types of liposomes and single walled
carbon nanotubes (SWCNTs). Liposomes are lipid vesicles containing
one or more concentric phospholipidic bilayers separated by hydro-
philic compartments. Such lipid particles have wide applicability in
biomedical field being used especially as membrane models and as
drug delivery systems to carry various therapeutic agents. The novelty
of this research consists in the way of design, preparation and character-
ization of new biohybrids based on biomimetic membranes, silver
phyto-nanoparticles (eco-synthesized using an aqueous leaf mint
extract) and carbon nanotubes (CNTs). Chlorophyll a inserted in lipo-
somes was used as an antioxidant agent and as a spectral marker to in-
vestigate the changes at molecular level in artificial lipid bilayers.
In our paper, the choice of Mentha piperita as a reducing agent for
biofabrication of silver nanoparticles is based on its wide bioavail-
ability and high phytotherapeutic potential. Recent studies showed
that M. piperita can be used in a wide variety of medicines, ranging
from pain relievers [13] to larvicide and mosquito repellent [14],
proving ancient people right when using M. piperita as medicine for
different affections such as motion sickness, seasickness, nausea,
diarrhea, vomiting, headaches and common cold [15].
The presented procedure involves four steps: 1) the phytosynthesis
of silver nanoparticles using an aqueous extract from mint leaves
trough a green strategy; 2) preparation of liposomes; 3) preparation
of liposome/mint–AgNP biohybrids and 4) preparation of liposome/
mint–AgNP/SWCNT biohybrids.
The resulting silver-based biomaterials presented antioxidant and
antimicrobial properties that have been considerable improved in the
presence of carbon nanotubes.
2. Materials and methods
2.1. Materials
Silver nitrate, KH2PO4, Na2HPO4, luminol (5-amino-2,3-dihydro-
phthalazine-1,4-dione), Tris (hydroxymethylaminomethane base),
HCl, and H 2 O 2 were purchased from Merck (Germany). Methanol
(99.9%), SWCNTs, sodium chloride and the lipids used for liposome
preparation: dipalmitoyl phosphatidylcholine (DPPC) and cholesterol
(Chol) were supplied from Sigma Aldrich (Germany).
The antibacterial activity was tested against human pathogenic
microbial strains such as Escherichia coli ATCC 8738, Staphylococcus
aureus ATTC 25923 and Enterococcus faecalis ATCC 29212. The bacte-
rial strains were grown in Luria Bertani Agar (LBA) plates at 37 °C
with following composition: peptone (Merck), 10 g/L; yeast extract
(Biolife) 5 g/L, NaCl (Sigma-Aldrich) 5 g/L and agar (Fluka) 20 g/L.
The stock culture was maintained at 4 °C. Cefotaxime used as a pos-
itive control in antimicrobial assay was supplied by Sandoz.
2.2. Sample preparation
2.2.1. Phytosynthesis of mint–silver nanoparticles
2.2.1.1. Preparation of mint extract. Dried mint (M. piperita L.) leaves sup-
plied by Fares Bio Vital (Romania) were washed in distilled water and
cut into small fine pieces (see Fig. 1). 50 g of this vegetal material was
transferred into a 500 mL Erlenmeyer flask containing 250 mL of dis-
tilled water and boiled for 5 min in order to release the intracellular ma-
terial into solution. The resulted aqueous mint leaf extract was cooled
and filtered through a filter paper in order to obtain a clear extract
(sample P1).
2.2.1.2. Synthesis of mint–silver nanoparticles. The herbal silver nanopar-
ticles (AgNPs) were prepared via a green route as previously de-
scribed [12]. 10 mL of mint extract was mixed with 10 mL of 1 mM
AgNO3 aqueous solution. This mixture was allowed to stay overnight
at room temperature. The phytosynthesis of mint–AgNPs (sample
P2) was firstly evidenced by visual inspection: the color of mixture
turned from pale brownish yellow to dark reddish brown accompa-
nied by a mirror like illumination on the walls of the flask due to ex-
citation of surface plasmon vibrations in nanoscaled silver particles.
The color changing of the medium is a proof of the plant extract
action as a bioreducing agent that reduces silver ions (Ag+ 0→ Ag0).
2.2.2. Biohybrid preparation
2.2.2.1. Liposome preparation. Thin film hydration method [16] was used
to obtain two kinds of multilamellar lipid vesicles (MLVs, 0.5 mM) with
and without cholesterol in the artificial lipid bilayers (DPPC/Chol molar
ratio = 4:1) which were suspended in a phosphate buffer solution (PB
PBS, KH2PO4–Na2HPO4 pH 7.4).
Chlorophyll a (Chla), a natural antioxidant porphyrin, was ex-
tracted from spinach leaves according to Strain and Svec method
[17]. Considering its antioxidant properties [18] and spectral features,
this photopigment was inserted (Chla/lipid molar ratio = 1/100)
into both types of liposomes: Chla–DPPC–MLVs (sample P3) and
Chla–Chol (20%)–DPPC–MLVs (sample P4) as described previously
[19].
2.2.2.2. Preparation of liposome/mint–AgNP biohybrids. The liposome/
mint–AgNP biohybrids (samples P5 and P6) were obtained by sonica-
tion. Appropriate amount of mint–AgNPs was added to MLV suspen-
sions (MLVs/AgNPs = 30/1, v/v) and the resulted mixtures were
subjected to an ultrasound treatment (Hielser titanium probe sonicator,
UP 100H).
2.2.2.3. Preparation of liposome/mint–AgNP/SWCNT biohybrids. A previ-
ously sonicated volume of a stock suspension of SWCNTs (0.9 mg/mL)
in PB PBS (pH 7.4) was added (in a final concentration of 0.05 mg/mL)
to a mixture composed of MLV suspension and mint–AgNPs (MLVs/
AgNPs = 30/1, v/v) and further sonicated resulting in liposomes/
mint–AgNPs/SWCNT biohybrids (samples P7 and P8).
Fig. 1 shows the preparation schema of the silver nanoparticles
and biohybrids (based on liposomes, mint–nanosilver and carbon
nanotubes) using an aqueous mint leaf extract. The sample codes
are resumed in Table 1.
In order to avoid the sample photodamage, all of the experiments
were carried out in dark.
2.2.3. Characterization methods
2.2.3.1. Absorption spectroscopy. Absorption spectra were recorded using
a double beam M400 Carl Zeiss Jena UV–VIS spectrophotometer in
200–800 nm range, operated at a resolution of 1 nm, with 1 nm slit
width and 0.3 nm/s scan rate.
 M.E. Barbinta-Patrascu et al. / Materials Science and Engineering C 39 (2014) 177–185
Fig. 1. Schematic representation of silver nanoparticle and biohybrid (based on liposomes, mint–nanosilver and carbon nanotubes) preparation using an aqueous mint extract.
2.2.3.2. ATR-FTIR spectroscopy. Fourier transformed IR (FTIR) spectral
analyses were performed on a Perkin Elmer Spectrum GX instrument
with attenuated total reflectance (ATR) diamond crystal, at a spectral
resolution of 4 cm− 1. For each spectrum, scans were accumulated in
the range of 400–4000 cm− 1.
2.2.3.3. Dynamic light scattering (DLS) measurements. The size of mint–
AgNPs given by the hydrodynamic diameters, Zaverage (the particle
diameter plus the double layer thickness) has been measured using
Dynamic Light Scattering technique (Zetasizer Nano ZS, Malvern Instru-
ments Ltd., U.K.), in the range between 0.6 nm and 6.0 μm, at 25 °C
temperature and at a scattering angle of 90°. The average diameters
(based on Stokes–Einstein equation) and the polydispersity indexes
(PdI, indicating the width of the size distribution) were obtained from
3 individual measurements using intensity distribution.
2.2.3.4. Scanning electron microscopy (SEM). Scanning electron micro-
scopic (SEM) analysis of the silver phytonanoparticles was done using
Table 1
The codes of the sample prepared.
Sample
Mint aqueous leaf extract
Mint–AgNPs
Chla–DPPC–MLVs
Chla–Chol–DPPC–MLVs
Chla–DPPC–MLVs/mint–AgNP hybrid
Chla–Chol–DPPC–MLVs/mint–AgNP hybrid
Chla–DPPC–MLVs/mint–AgNPs/CNT hybrid
Chla–Chol–DPPC–MLVs/mint–AgNPs/CNT hybrid
179
a FEI Quanta 200 Scanning Electron Microscope in a low vacuum work-
ing mode. One drop of mint–AgNPs placed on an aluminum support was
left to dry overnight. The SEM images were taken using a secondary
electron detector.
2.2.3.5. X-ray fluorescence (XRF). The X-ray fluorescence technique was
used to detect silver presence in the samples. The XRF determinations
have been carried out in Helium atmosphere, without any filter, for a
period of 300 s, at proper voltage and current intensity by using a
3.6 μm Mylar tissue. A PW4025-MiniPal-Panalytical type energy disper-
sive XRF spectrometer with a rhodium anode was used in these
experiments.
2.2.3.6. Chemiluminescence (CL) assay. The in vitro antioxidant activity
of the samples has been determined by chemiluminescence (CL)
assay using a Chemiluminometer Turner Design TD 20/20, USA. The
oxidative degradation of luminol occurred in the presence of H2O2
in alkaline buffer (pH = 8.6), generates a wide range of free radicals
of oxygen.
The antioxidant activity (AA%, as percentage of free radical scaveng-
ing) of each sample was obtained using the mathematical expression:
AA ¼ ½ðI 0 −IÞ=I 0   100% ð1Þ
Code
P1 where I0 is the maximum CL intensity for standard at t = 5 s and I is the
P2
maximum CL intensity for sample at t = 5 s [20].
P3
P4
P5 2.2.3.7. Antibacterial assay. Antibacterial assay was performed by agar
P6 well diffusion method [21,22]. Sterile LBA plates were prepared by
P7 pouring the sterilized media in sterile Petri dishes under aseptic condi-
P8
tions. 1 mL of the test microorganism was spread on agar plates. Using a
180 M.E. Barbinta-Patrascu et al. / Materials Science and Engineering C 39 (2014) 177–185
sterile Durham tube 6 mm diameter, the wells were made according
to the number of samples. The wells were inoculated with 50 μL of
sample.
Dispersion media S1 (distilled water) and S2 (phosphate buffered
solution, PB PBS) serve as negative controls. Cefotaxime (2 mg/mL)
in PB PBS or in distilled water was prepared as a positive control.
Cefotaxime (a cephalosporin-type antibiotic) works by interfering
with the ability of bacteria to form cell walls. Cefotaxime is a
broad-spectrum antibiotic that kills a wide variety of bacteria that
cause a wide variety of commonly-occurring infections [23].
All of the plates containing bacteria were incubated in the Laboshake
Gerhardt thermoshaker at 37 °C for 24 h. Antibacterial activity of the
samples against microorganism species was determined by measuring
the size of inhibition zone (IZ, mm) as a clear, distinct zone of growth in-
hibition surrounding agar wells, and values b 8 mm were considered as
not active against microorganisms.
The percentage growth inhibition was calculated by the relation:
Percentage inhibitionð% Þ ¼ ðTS−DCÞ=PC  100
where TS, DC and PC represents the inhibition zone of test sample, of
dispersant control and of positive control, respectively [23].
All of the experiments were performed in triplicate. The results
are reported as the average of three experiments and are presented
as mean ± standard deviation (SD). Standard deviation was calcu-
lated as the square root of variance using STDEV function in Excel
2010.
2.2.3.8. Atomic force microscopy (AFM). AFM images were recorded on
Integrated Platform SPM-NTegra model Prima in semi-contact mode
(scanning area had a range of 1.2 × 1.2 μm2) using a NSG01 cantilever
with a typical curvature radius of 10 nm. All AFM measurements were
obtained on a mica substrate. The top layer of mica was peeled off and
a small volume of sample was applied onto a fresh mica layer and left
to dry overnight.
3. Results and discussion
3.1. Characterization of silver nanoparticles phytosynthesized using a mint
extract
The biosynthesis of mint–AgNPs was confirmed by ATR-FTIR and
absorption spectroscopy. Aqueous extract from M. piperita leaves
acts both as reducing and capping agent in silver nanoparticle
phytosynthesis. The particle size was evaluated by DLS measurements
and the morphological aspects of these silver phytonanoparticles were
obtained by SEM analysis.
3.1.1. Characterization of herbal silver nanoparticles by absorption
spectroscopy
The phytosynthesis of metal nanoparticles was evaluated by UV–VIS
absorption spectroscopy.
The SPR (surface plasmon resonance) band detected in the mint–
AgNP spectrum, around 450 nm (Fig. 2), is due to excitation of Surface
Plasmon Vibrations in metallic nanoparticles synthesized using the
aqueous mint extract. This is a signature of the biosynthesis of monodis-
perse and spherical-shaped herbal silver nanoparticles, results further
confirmed by SEM micrographs and DLS measurements.
It could be noticed that the plant extract displayed no absorption in
the spectral window between 400 and 550 nm.
3.1.2. Characterization of mint–silver nanoparticles by ATR-FTIR
spectroscopy
Fourier transform infrared spectroscopy (FTIR) was used to predict
the involvement of functional groups in silver bioreduction. Individual
Fig. 2. The absorption spectra of mint extract and mint–AgNPs.
ð2Þ
ATR FTIR spectra were recorded for AgNO3 solution, M. piperita extract
and mint–AgNPs and presented comparatively in Fig. 3.
These spectra illustrate strong broad IR bands at 3360 cm−1 (in mint
extract), 3256 cm− 1 (in mint–AgNPs) and a weak broad band at
3439 cm−1 (in silver nitrate solution) corresponding to O\H stretching
(hydrogen-bonded).
The two bands at 2868 cm− 1 and 2980 cm− 1 present in plant
extract are characteristic for C\H asymmetric and symmetric
stretching vibrations of saturated C (sp 3), respectively. After re-
duction of silver ions, only a single C\H stretching (alkyls) IR band
(at 2935 cm− 1) was present. Mint extract exhibited weak IR bands
at 1142, 1075 and 1046 cm− 1 assigned to C\N stretching vibrations
of aliphatic amines or C\O stretching vibrations of alcohols/phenols,
which are originated from different phytoingredients present in the
plant extract (polyphenols, polysaccharides and proteins). The mint
extract FTIR spectrum revealed a weak band at 1716 cm− 1 attributed
to amide I (arising from stretch vibration of carbonyl C_O of proteins)
and a strong sharp band at 1353 cm−1 (arising from the C\O group of
polyols like catechins or hydroxyflavones) [24–29]. Instead of the weak
band at 1611 cm−1 (assigned to amide I, arising due to carbonyl stretch
in proteins) in mint extract spectrum, a strong sharp band at 1594 cm−1
(corresponding to C_O stretching) appeared in herbal nanosilver spec-
trum. The weak band at 1513 cm−1 due to C_C stretching associated
with the aromatic skeletal mode [25] of the mint extract is shifted to
1526 cm−1 after addition of AgNO3 solution.
The sharp bands occurred at 801 cm−1 and at 825 cm−1 present in
the silver nitrate spectrum disappeared when mint–AgNPs are formed,
whilst the strong sharp band at 1384 cm−1 in AgNO3 spectrum, attrib-
uted to symmetric stretching vibrations of the nitrate group [30], is
very weakened and shifted to 1399 cm− 1 in spectrum of silver
phytonanoparticles. These observations indicate that silver nanoparti-
cles are biosynthesized after addition of mint extract over the AgNO3
solution.
ATR-FTIR results demonstrated the involvement of biomolecules
(carbohydrates, aminoacids, proteins) and of the phytoingredients con-
taining aldehyde and hydroxyl functional groups (polyphenols) units in
Ag+ bioreduction by mint extract. The Ag+ ions arising from silver
nitrate solution accepts electrons from the phytochemicals present in
M. piperita extract resulting in their phytoreduction.
3.1.3. Size determination and morphological characterization of silver
phytonanoparticles
Dynamic light scattering (DLS) technique was used to evaluate the
dimension of herbal silver nanoparticles. The particle size distribution
profile of mint–AgNPs (Fig. 4) exhibited an average diameter of 75 nm
 M.E. Barbinta-Patrascu et al. / Materials Science and Engineering C 39 (2014) 177–185
Fig. 3. ATR-FTIR spectra of aqueous mint extract, mint–AgNPs and aqueous solution of 1 mM AgNO3.
and a good polydispersity index (PdI = 0.19) indicating the presence of
monodispersed particles.
SEM analysis confirmed the phytosynthesis of nano-scaled silver
particles and provided useful morphological details regarding mint–
AgNPs. SEM micrograph (Fig. 5) displays high density of spherical
shaped silver nanoparticles, well distributed.
3.2. Characterization of silver phytonanohybrids
The resulted silver biohybrids were characterized by different
techniques: X-ray fluorescence analysis, chemiluminescence assay,
antimicrobial activity, VIS absorption spectroscopy and atomic
force microscopy.
3.2.1. XRF analysis of M. piperita silver biohybrids
X-ray fluorescence technique was used in order to check the silver
presence in the prepared bioconstructs. XRF analysis (Fig. 6) confirmed
the existence of silver in the samples P5, P6, P7 and P8, so the formation
of biohybrids based on mint–AgNPs, liposomes and/or carbon nano-
tubes. As expected, the liposomes P3 and P4 used as references did
not contain silver.
3.2.2. Antioxidant properties of the samples
All of the samples were subjected to an oxidative stress simulated
in vitro and the antioxidant activity (AA%) values were calculated
(Fig. 7) according to relation (1) (see Section 2.2.3.6).
The liposomes showed weak antioxidant activity values (26.4%
for Chla–DPPC–MLVs and 32% for Chla–Chol–DPPC–MLVs), while
Fig. 4. Particle size distribution of mint–AgNPs.
181
the rest of the samples presented elevated values. The presence of
cholesterol in lipid bilayers led to a moderate increase in the AA%
values of Chla–liposomes.
A significant enhancement of antioxidant activity (69.7% for
Chla–DPPC–MLVs and 62.8% for Chla–Chol–DPPC–MLVs) occurred
in the case of addition of herbal silver nanoparticles to these lipid vesicle
suspensions (AA = 87% for P5 and AA = 86% for P6 biohybrids).
CNTs addition to silver phyto-AgNPs/liposome bioconstructs
increased the antioxidant activity values to 87% and 86% for P5 and,
respectively, P6 biohybrids.
SWCNT-based bioconstructs exhibited antioxidant activity values
(91% for P7 and 90.8% for P8 hybrids) greater than silver phyto-
AgNPs/liposome biohybrids. In the last decade, scientists reported
antioxidant properties of carbon nanotubes [31–33]. Fenoglio et al.
(2006) observed that CNTs abolished the cytochrome c oxidation
by superoxide radicals and supposed that the potential capacity of
CNTs as free-radical scavengers is probably due to their high electron
affinity. Carbon nanotubes can trap the free radicals by grafting them
Fig. 5. SEM micrograph of the mint–silver nanoparticles.
182 M.E. Barbinta-Patrascu et al. / Materials Science and Engineering C 39 (2014) 177–185

Fig. 6. XRF investigation of silver presence in the biohybrids P5, P6, P7 and P8 (the absence of silver in the samples P3 and P4 used as references is observed).

at the CNT surface, via radical addition to the carbon framework, thus
decreasing the oxidative stress [34].
The aqueous mint leaf extract have strong antioxidant properties
(AA = 93.8%) due to polyphenolic compounds especially and also
other active phytochemicals occurred in M. piperita extract [35].
The silver phyto-nanoparticles (P2) exhibited better antioxidant ac-
tivity (AA = 97%). Even in small quantity (3.2%), the mint–AgNPs
confer high antioxidant properties to biohybrids.
The strong antioxidant activity values of carbon-based silver
phytohybrids (91% and 90.8% for P7 and P8 hybrids, respectively)
make them good candidates in biomedical applications to fight against
harmful effects of free radicals.
3.2.3. Antimicrobial properties of mint–silver nanostructures
The antimicrobial investigations were performed on both Gram-
negative (E. coli) and Gram-positive (S. aureus and E. faecalis) bacteria
(Table 2).
Distilled water (S1) serves as a negative control for mint extract
(P1) and silver nanoparticles (P2). Phosphate buffer solution pH
7.4 (S2) is the negative control for liposomes (P3 and P4) and
biohybrids (P5, P6, P7, P8). Liposomes alone showed non significant
antibacterial activity.
Mint has strong antimicrobial properties and its vapors are believed
to prevent the spread of infectious diseases [36]. As expected, the aque-
ous M. piperita extract exhibited good biocidal effect against all bacteria

Table 2
Zone of inhibition (mm; in diameter) against different bacterial strains by eco-designed
biohybrids.

Sample/(control)⁎ IZ (mm) for microorganisms used

Escherichia coli Staphylococcus aureus Enterococcus faecalis

S1 0 0 0
S2 5.3 ± 0.057 5.1 ± 0.011 2.0 ± 0.000
Cefotaxime/(S1) 33.3 ± 0.030 33.5 ± 0.000 29.0 ± 0.060
Cefotaxime/(S2) 36.0 ± 0.000 35.0 ± 0.200 33.2 ± 0.040
P1/(S1) 8.2 ± 0.069 8.1 ± 0.057 8.0 ± 0.000
P2/(S1) 25.3 ± 0.057 15.4 ± 0.069 21.0 ± 0.100
P3/(S2) 5.7 ± 0.069 5.6 ± 0.057 4.9 ± 0.069
P4/(S2) 6.1 ± 0.069 5.9 ± 0.057 5.0 ± 0.000
P5/(S2) 12.0 ± 0.011 12.0 ± 0.057 10.0 ± 0.069
P6/(S2) 13.3 ± 0.057 11.3 ± 0.200 10.3 ± 0.057
P7/(S2) 16.0 ± 0.069 15.0 ± 0.057 13.0 ± 0.200
P8/(S2) 25.0 ± 0.100 23.0 ± 0.069 23.0 ± 0.100
⁎ First column presents the controls and the sample codes accompanied by their
dispersants (S1 or S2) in the brackets. The negative controls are distilled water (S1)
Fig. 7. Antioxidant activity values of the mint extract (P1), mint–AgNPs (P2), liposomes and phosphate buffer solution (S2); the positive control is Cefotaxime (2 mg/mL)
(P3, P4) and biohybrids (P5, P6, P7, P8). dissolved in S1 or S2 (see Section 2.2.3.7).
 M.E. Barbinta-Patrascu et al. / Materials Science and Engineering C 39 (2014) 177–185 183

tested and the silver nanoparticles synthesized using this plant extract S. aureus strain, enhanced from 11 mm (see sample P6) to 23 mm
showed very high antimicrobial activity (see Table 2). (see sample P8).
As is well known, Gram-positive bacteria were much more difficult A synergistic effect occurred when silver nanoparticles and car-
to destroy, due to the more complicated cell wall structure consisting bon nanotubes were simultaneously added to liposomes resulting
of several layers of peptidoglycan which are thicker as compared to in biohybrids with better antibacterial properties.
Gram-negative bacteria those cell wall is relatively thin and composed The maximum in vitro inhibition of tested microorganisms was
of a single peptidoglycan layer [37]. Despite this fact, the mint extract scored in Chla–DPPC–Chol–MLVs/mint–AgNPs/SWCNT biocomposites
and the silver-based biohybrids were highly effective against all of the (sample P8) which offered inhibition zone of 25 mm against E. coli
bacterial strains. and 23 mm against both S. aureus and E. faecalis (Table 2).
The process for the synthesis of mint nanoparticles and biocomposites Fig. 8 shows the comparative percent inhibition of all samples
(based on liposomes, mint–nanosilver and carbon nanotubes) in against the three tested bacterial strains. The percentage growth in-
large scale using this readily available plant extract may have com- hibition was calculated with relation (2) presented in Section 2.2.3.7
mercial viability and open new opportunities to develop studies in using Cefotaxime (dissolved in distilled water or in PB PBS) as a pos-
the interface between biology and material science. itive control. Excepting liposomes, all of the samples were highly
Even in small quantity (MLVs/AgNPs = 30/1, v/v), the mint–silver active against the tested microorganisms.
nanoparticles confer strong biocidal properties to biohybrids. The Silver-based biocomposites P5, P6 and P7 exhibited moderate
presence addition of CNTs (0.05 mg SWCNTs/mL liposomal suspen- activities against all strains in a range of 17.7–33.1% inhibition. No
sion) into to hybrid phytostructures enhanced the antimicrobial significant differences concerning the inhibition of the three tested
activity of mint–silver nanoparticles/liposome biocomposites. Car- microorganisms were observed at the biohybrids P5 and P6. Among
bon nanotubes possess antimicrobial properties [38,39] and small silver-based biohybrids, only the Chla–DPPC–Chol–MLVs/mint–
amounts of SWCNTs were enough to achieve high antimicrobial ac- AgNPs/SWCNT biocomposites (sample P8) showed good inhibition
tivity. The high CNT potency against bacteria could be explained by of all tested bacterial strains (more than 50%). Between all samples,
an efficient contact with the surface of bacteria cell by hydrophobic the biohybrid P8 showed maximum inhibition of S. aureus (51.1%).
interactions resulting in perturbation of bacterial cell integrity and
then cell membrane damage. Scientists demonstrated that cell mem-
brane damage resulting from direct contact with SWCNTs is the likely
mechanism leading to bacterial cell death [40–42]. The mechanism
of antimicrobial activity of SWCNTs is associated also with their
diameter-dependent piercing and length-dependent wrapping on
the lysis of bacterial walls and membranes, inducing release of intra-
cellular components (DNA and RNA) and allowing a loss of bacterial
membrane potential, demonstrating complete destruction of micro-
organism [43–45]. Deokar et al. found that the antibacterial activity
is attributed also to a molecular-scale interaction with surface func-
tional groups of bacteria [46].
Bio-inspired materials based on biomimetic lipid bilayers, phyto-
AgNPs and SWCNTs have the ability to better interact with bacteria
cell by means of the liposomal component that facilitates a tight contact
between bacteria and biohybrids. Artificial lipid bilayers mimicking the
biomembranes can readily fuse with bacterial cell membrane [47].
No significant differences regarding biocidal effect were observed at
the silver-based biocomposites P5 and P6. In the case of cholesterol-
containing biohybrids, addition of carbon nanotubes had a considerable
effect, therefore the growth inhibition was noted (Table 2). Thus, for
example, in the presence of CNTs, the inhibition zone diameter for

Fig. 9. Absorption spectra of liposome-based materials without cholesterol (a) and with
Fig. 8. Comparative percent inhibition of all samples against different bacterial strains. cholesterol (b).
184 M.E. Barbinta-Patrascu et al. / Materials Science and Engineering C 39 (2014) 177–185
3.2.4. Characterization of silver-based biohybrids by VIS absorption
spectroscopy
Fig. 9 displays the VIS absorption spectra of the samples, which
were normalized against the absorption at the maximum in red of
Chla embedded in artificial lipid bilayers. These spectra show the
“over-estimated absorbances” due to the light scattering induced
by AgNPs in samples P5 and P6 and by AgNPs and CNTs in samples
P7 and P8.
The modifications in spectral features of Chla incorporated in
liposomes without Chol (Fig. 9a) are more pronounced than in
samples with Chol (Fig. 9b) and the position of the main red peak
(672 nm) of Chla was shifted by 2 and 5 nm (Fig. 9a). As already
known, cholesterol enhances the lipid membrane stability [48,49],
thus in the case of cholesterol-containing samples, the position of
the Chla absorption maximum did not significantly changed, so the
Chla location in the Chol–biomimetic membranes is maintained ap-
proximately in the same place (671 nm).
The modifications occurred in the Soret band showed that dimen-
sions and the structure of liposomes are affected in the presence of silver
nanoparticles and carbon nanotubes.
3.2.5. AFM analysis of the liposomes/mint–AgNPs/SWCNT hybrids
The silver-based biohybrids with better antimicrobial properties
(sample P8: Chla–DPPC–Chol/mint–AgNPs/SWCNTs) were subjected
to AFM investigation that revealed the 3-D architecture and the
morphological aspects of these silver-based materials (Fig. 10). The
AFM images show the noncovalent functionalization of SWCNTs by
their decoration with liposomes and mint–AgNPs. A lipid coating ob-
served around the carbon nanotubes that prevents their aggregation
may confer biocompatibility of SWCNTs. SWCNTs form a network of
nanotubes interconnected by artificial lipid membranes. Spherical
Fig. 10. AFM micrographs of Chla–DPPC–Chol/mint–AgNPs/SWCNT biohybrids in height
(a) and 3D representation (b).
and quasi-spherical shaped profiles of liposomes, mint–AgNPs and
liposomes/phyto-AgNPs were observed along and near the carbon
nanotubes.
4. Conclusions
This paper described an original green bottom-up method to build
noncovalent entities (based on lipid vesicles, phytonanosilver and car-
bon nanotubes) with antioxidant and antimicrobial properties.
Phytosynthesis of AgNPs using the Mentha extract combines the
benefits of this herb with the interesting properties of silver.
In this work two kinds (with and without cholesterol) of lipid vesi-
cles have been developed. Chla embedded into liposomes provided use-
ful information regarding the changes occurred in artificial lipid
bilayers. The results obtained by monitoring the absorption spectra of
biohybrids using this phytopigment as a spectral marker, revealed the
existence of interactions between silver nanoparticles and carbon nano-
tubes with the biomimetic membranes, with significant effects on the
structure of the lipid bilayers.
The presence of CNTs in the silver-based biohybrids resulted in
biocomposites with better properties, thus the eco-designed
bioconstructs consisting of Chol–liposomes, mint–nanosilver and
carbon nanotubes exhibited high power of free radicals scavenging
(AA = 90.8%) and have been shown to be strong biocides offering
inhibition zone of 25 mm against E. coli and 23 mm against S. aureus
and E. faecalis. The Chla–DPPC–Chol–MLVs/mint–AgNPs/SWCNT
biocomposite (sample P8) was the most effective against S. aureus
(51.1% inhibition).
The building of bionanocomposites based on biomimetic mem-
branes, silver phytonanoparticles and carbon nanotubes presented
in this study, opens new perspectives for biomedical (nanovectors,
topical use) and biotechnological (antioxidant and antimicrobial
coating materials) applications.
Acknowledgments
This work was supported by the strategic grant POSDRU/89/1.5/S/
58852, Project “Postdoctoral programme for training scientific
researchers” cofinanced by the European Social Fund within the Secto-
rial Operational Program Human Resources Development 2007–2013.
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