molecules
Article
Antimicrobial Lemongrass Essential Oil—Copper
Ferrite Cellulose Acetate Nanocapsules
Ioannis L. Liakos 1, *, Mohamed H. Abdellatif 2 , Claudia Innocenti 3 , Alice Scarpellini 4 ,
Riccardo Carzino 1 , Virgilio Brunetti 5 , Sergio Marras 4 , Rosaria Brescia 4 , Filippo Drago 4
and Pier Paolo Pompa 6
1
2
3
4
5
6
*
Smart Materials Group, Nanophysics Department, Istituto Italiano di Tecnologia (IIT), Via Morego 30,
16163 Genoa, Italy; Riccardo.Carzino@iit.it
Nanostructures Department, Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163 Genoa, Italy;
Mohamed.Abdellatif@iit.it
INSTM-RU of Florence and Department of Chemistry, University of Florence, Via Della Lastruccia 3-13,
50019 Sesto F.no, Firenze, Italy; claudia.innocenti@unifi.it
Nanochemistry Department, Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163 Genoa, Italy;
Alice.Scarpellini@iit.it (A.S.); Sergio.Marras@iit.it (S.M.); Rosaria.Brescia@iit.it (R.B.);
Filippo.Drago@iit.it (F.D.)
Biomolecular Nanotechnologies, Istituto Italiano di Tecnologia (IIT), Via Barsanti 1, 73010 Lecce, Italy;
Virgilio.Brunetti@iit.it
Nanobiointeractions & Nanodiagnostics, Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163 Genoa,
Italy; Pierpaolo.Pompa@iit.it
Correspondence: ioannis.liakos@iit.it; Tel.: +39-107-178-1705
Academic Editor: Alexandru Mihai Grumezescu
Received: 19 February 2016; Accepted: 14 April 2016; Published: 20 April 2016
Abstract: Cellulose acetate (CA) nanoparticles were combined with two antimicrobial agents,
namely lemongrass (LG) essential oil and Cu-ferrite nanoparticles. The preparation method of
CA nanocapsules (NCs), with the two antimicrobial agents, was based on the nanoprecipitation
method using the solvent/anti-solvent technique. Several physical and chemical analyses were
performed to characterize the resulting NCs and to study their formation mechanism. The size
of the combined antimicrobial NCs was found to be ca. 220 nm. The presence of Cu-ferrites
enhanced the attachment of LG essential oil into the CA matrix. The magnetic properties of the
combined construct were weak, due to the shielding of Cu-ferrites from the polymeric matrix, making
them available for drug delivery applications where spontaneous magnetization effects should be
avoided. The antimicrobial properties of the NCs were significantly enhanced with respect to CA/LG
only. This work opens novel routes for the development of organic/inorganic nanoparticles with
exceptional antimicrobial activities.
Keywords: antimicrobial nanocapsules; lemongrass essential oil; copper ferrite nanoparticles;
cellulose acetate; drug delivery; antimicrobial resistance
1. Introduction
Cellulose acetate (CA) is a biopolymer prepared through the reaction of cellulose with acetic
anhydride and acetic acid in the presence of sulfuric acid [1]. Cellulose acetate has many applications
in the pharmaceutical field, including drug or enzyme delivery [1],wound dressings of CA nanofibers
with essential oils [2], chlorinated CA with modified 4,41 -diphenylmethane diisocyanate nanofiber
mats with antimicrobial properties [3], electrospun CA with other polysaccharide nanofibers [4] with
biomedical properties, antifouling coatings of CA thin films with polysaccharide multilayers [5] and
others. During the last few years CA nanoparticles have been formed [6] and incorporated with
Molecules 2016, 21, 520; doi:10.3390/molecules21040520
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Molecules 2016, 21, 520
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chitosan, which has antimicrobial properties [7]. Recently, pure CA nanocapsules (NCs) loaded with
natural antimicrobial agents, such as essential oils, have been prepared with considerable antimicrobial
efficiency [8]. Essential oils are well known for their antimicrobial properties [9] and for being kept
active, even if encapsulated into polymers [2,10,11].
Magnetic nanoparticles, such as iron oxide nanoparticles, have been found to be effective
in fighting infectious diseases [12–15]. Transition metals of copper, zinc, chromium and nickel
have been substituted into cobalt ferrite nanoparticles and have shown very good antimicrobial
properties [16]. Moreover, silver nanoparticles loaded into copper ferrite (CuFe2 O4 ) magnetic hollow
fibers showed excellent antimicrobial efficacy against four bacteria: E. coli, S. typhi, S. aureus and
V. parahaemolyticus [17].
Ferrites are semiconductor ceramics with the general formula of spinel structure AB2 O4 , where
A and B represent various metal cations, including iron. Ferrites are considered a class of spinels
that consist of cubic closed pack oxides with A cations occupying 1/8th of the octahedral sites, and B
cations occupying half of the octahedral sites. For the inverse spinel structure, half of the B cations
occupy tetrahedral sites, and both the A and B cations occupy the octahedral sites. Meanwhile, divalent
and trivalent cations can occupy both A and B sites [18–20]. Cu-ferrite is known to have non-collinear
saturation magnetization, due to dilution of the magnetic properties with Cu ions [21].
Some kind of ferrites, such as Cu-ferrites, are of great interest as soft magnets, since they
can be magnetized, de-magnetized and their magnetic properties controlled through encapsulation.
Some researchers suggested that substitution of spinel iron oxide with metals can help in enhancing
the biomedical properties of the ferrite nanoparticles [16,22].
In this work, we studied the effect of substituting the spinel iron oxide by Cu ions to form
Cu-ferrite with low copper content. Exploiting the nontoxicity of Cu-ferrites [23], we combined copper
ferrite of the spinel structure, CuFe2 O4 , prepared by the co-precipitation method, with CA/lemongrass
(LG) NCs to improve their antimicrobial activity. The Cu-ferrite nanoparticles considerably enhanced
the antimicrobial activity of CA/LG essential oil NCs and showed complete block of bacteria growth.
The final nanocomposite structure of CA/LG/Cu-ferrite NCs showed significant enhancement in the
antimicrobial properties compared to the previous study of CA/LG NCs alone [8].
2. Results and Discussion
2.1. Dynamic Light Scattering
DLS was used to determine the diameter of the formed nanoparticles and nanocapsules. It was
found that the addition of either Cu-ferrite NPs or LG essential oil lowered the size of the CA
NPs (440 nm) (Figure 1). The size of the CA/Cu-ferrite NCs was 340 nm, whereas the size of
the CA/5-LG NCs was 150 nm (Figure 1). By adding both antimicrobial agents, the overall size
of the CA/5-LG/Cu-ferrite NCs was about 225 nm (Figure 1), higher than the CA/5-LG NCs
(150 nm) and bare Cu-ferrite NPs (70 nm), as will be shown later in the SEM section, revealing
that Cu-ferrite increased the size of the CA/5-LG NCs. The polydispersity index (PdI) of the CA/5-LG
and CA/5-LG/Cu-ferrite NCs with the presence of LG was low, whereas the PdI index of CA NPs and
CA/Cu-ferrite NCs was high, revealing that LG essential oil helped to stabilize the formation of the
resulting NCs, as shown in Figure 1. Similarly, and for the same reason, the intensity % is higher when
LG essential oil was present. The reason that the Cu-ferrite and LG oil lowers the diameter of bare CA
was due to the reaction between them and the consequent encapsulation, as will be demonstrated later,
making them more stable than bare CA NPs.
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500
400
400
300
0.6
0.6
100
96.4
439.8
100
0.6
100
0.6
0.5
100
90
0.5
0.4
0.42
439.8
337.4
0.42
0.4
0.3
337.4
222.1
300
200
200
100
96.4
PdI
Z-average
(d. nm)
Z-average
(d. nm)
600
500
PdI
600
148.5
60.9
CA60.9
60.8
0.14
148.5
CA/5-LG
0.14
0.198
222.1
0.198
60.8
CA/0.1-Cuferrite
CA/5-LG/0.1-Cuferrite
0.3
0.2
CA/5-LG
90
80
80
70
70
60
0.2
0.1
Sample
CA
Intensity %
Intensity %
Z-average (d. nm)
PdI
Z-average
Intensity % (d. nm)
PdI
Intensity %100
60
0.1
CA/0.1-Cuferrite CA/5-LG/0.1-Cuferrite
Sample
Figure
1. DLS
measurements ofof the
the size,
size, polydispersity
polydispersity index
(PdI)
andand
intensity
% of%the
Figure
1. DLS
measurements
index
(PdI)
intensity
of the
Cellulose
acetate
(CA)
nanoparticles,
CA/5-lemongrass
(LG),
CA/Cu-ferrite
and
CA/5-LG/Cu-ferrite
Cellulose
acetate
(CA)
nanoparticles,
(LG), CA/Cu-ferrite
andintensity
CA/5-LG/Cu-ferrite
Figure
1. DLS
measurements
of CA/5-lemongrass
the size, polydispersity
index (PdI) and
% of the
nanocapsules (NCs).
nanocapsules
(NCs).
Cellulose acetate (CA) nanoparticles, CA/5-lemongrass (LG), CA/Cu-ferrite and CA/5-LG/Cu-ferrite
(NCs).
2.2. nanocapsules
X-ray Diffraction
2.2. X-ray Diffraction
The Diffraction
bare Cu-ferrite NPs, investigated via X-ray diffraction (XRD), consist of a mixture of four
2.2. X-ray
The
barecrystalline
Cu-ferrite
NPs, as
investigated
via X-ray
diffraction (XRD),
consist
mixture
of four
different
phases,
shown in Figure
2. Semi-quantitative
analysis
based of
onathe
reference
The
bare
Cu-ferrite
NPs,
investigated
via
X-ray
diffraction
(XRD),
consist
of
a
mixture
of
four
different
crystalline
phases,
as shown
Figure 2.the
Semi-quantitative
analysis
based
on the
intensity
ratio (RIR)
was carried
out in
to estimate
amount of all of the
identified
phases,
withreference
the
different
crystalline
phases,
as
shown
in
Figure
2.
Semi-quantitative
analysis
based
on
the
reference
following
66 wtcarried
% tetragonal
2O4 (copper
ferrite, of
space
I41/amdS(141)),
24 wt
% the
intensity
ratio results:
(RIR) was
out toCuFe
estimate
the amount
all group:
of the identified
phases,
with
intensity
ratio
was carried
outgroup:
to estimate
the amount
of%all
of the2 (delafossite,
identified phases,
with
cubic
CuFe
2O(RIR)
4 (cuprospinel,
space
Fd-3m(227)),
6
wt
CuFeO
space
group:
following results: 66 wt % tetragonal CuFe2 O4 (copper ferrite, space group: I41/amdS(141)), 24the
wt %
following results:
66 %
wtCuO
% tetragonal
CuFe
O
4 (copper
ferrite,
space group:
I41/amdS(141)), 24NCs
wt %
4 wt
(tenorite,
space2Fd-3m(227)),
group:
Cc(9)).
XRD
on CA/5-LG/Cu-ferrite
cubicR-3m(166))
CuFe2 O4 and
(cuprospinel,
space
group:
6 wt analysis
% CuFeO
2 (delafossite, space group:
cubic
CuFe2O
4 (cuprospinel,
group: Fd-3m(227)),
wt to
% see
CuFeO
2 (delafossite,
space
group:
was mainly
covered
from the space
CA polymers,
and it was not6easy
the Cu-ferrite
NPs due
to their
R-3m(166)) and 4 wt % CuO (tenorite, space group: Cc(9)). XRD analysis on CA/5-LG/Cu-ferrite NCs
R-3m(166))
and 4 wt %
CuOthe
(tenorite,
space
group:
Cc(9)).(i.e.,
XRD
analysis
small concentration
below
sensitivity
of the
technique
below
1%).on CA/5-LG/Cu-ferrite NCs
was was
mainly
covered from the CA polymers, and it was not easy to see the Cu-ferrite NPs due to their
mainly covered from the CA polymers, and it was not easy to see the Cu-ferrite NPs due to their
small
concentration
below
thethe
sensitivity
below1%).
1%).
small concentration
below
sensitivityofofthe
thetechnique
technique (i.e.,
(i.e., below
Figure 2. XRD pattern of Cu-ferrite NPs.
2.3. Atomic Force Microscopy
Figure 2. XRD pattern of Cu-ferrite NPs.
2. XRD
pattern
Cu-ferrite NPs.
Atomic force microscopyFigure
was used
to reveal
theoftopographical
differences with the addition of
Cu-ferrite
NPs
in
the
nanocapsules.
As
was
observed
from
Figure
3a,
CA/5-LG NCs are spherical in
2.3. Atomic Force Microscopy
shape and
have
diameters less than 200 nm, as was also proven previously by DLS measurements.
2.3. Atomic
Force
Microscopy
Atomic force microscopy was used to reveal the topographical differences with the addition of
Atomic
microscopy
was used
reveal
the topographical
with
addition
Cu-ferriteforce
NPs in
the nanocapsules.
As to
was
observed
from Figure 3a,differences
CA/5-LG NCs
arethe
spherical
in of
Cu-ferrite
NPs
in
the
nanocapsules.
As
was
observed
from
Figure
3a,
CA/5-LG
NCs
are
spherical
shape and have diameters less than 200 nm, as was also proven previously by DLS measurements. in
shape and have diameters less than 200 nm, as was also proven previously by DLS measurements.
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When
NCs, the
the NCs
NCs were
were no
no longer
longer
When Cu-ferrite
Cu-ferrite was
was added
added to
to the
the NC,
NC, such
suchas
asthe
theCA/5-LG/Cu-ferrite
CA/5-LG/Cu-ferrite NCs,
spherical
and
resemble
more
the
shapes
of
Cu-ferrites,
i.e.,
tetragonal
and
cubic,
as
shown
in
Figure
3b.
spherical and resemble more the shapes of Cu-ferrites, i.e., tetragonal and cubic, as shown in Figure 3b.
Furthermore, the
that
thethe
CA/5-LG/Cu-ferrite
were
larger
in
Furthermore,
the 3D
3Dtopography
topographyininFigure
Figure3b3breveals
reveals
that
CA/5-LG/Cu-ferriteNCs
NCs
were
larger
size
than
the
NCs
without
Cu-ferrite
(Figure
3a),
reaching
sizes
of
more
than
200
nm,
in
accordance
in size than the NCs without Cu-ferrite (Figure 3a), reaching sizes of more than 200 nm, in accordance
with
with the
the DLS
DLS measurements
measurements shown
shown in
in Figure
Figure 1.
1.
Figure 3. AFM topography of (a) CA/5-LG NCs and (b) CA/5-LG/Cu-ferrite NCs.
Figure 3. AFM topography of (a) CA/5-LG NCs and (b) CA/5-LG/Cu-ferrite NCs.
2.4. High Resolution Scanning Electron Microscopy, Energy Dispersive X-ray Spectroscopy and
2.4. High Resolution
Scanning
Electron Microscopy, Energy Dispersive X-ray Spectroscopy and Transmission
Transmission
Electron
Microscopy
Electron Microscopy
High resolution scanning electron microscopy revealed the presence of the Cu-ferrite nanoparticles
High resolution scanning electron microscopy revealed the presence of the Cu-ferrite nanoparticles
and their distribution within the CA/5-LG NCs. The polygonal appearance of the Cu-ferrite nanoparticles
and their distribution within the CA/5-LG NCs. The polygonal appearance of the Cu-ferrite
is clearly shown in the inset in Figure 4a, which highlights also their tendency to aggregate in clusters,
nanoparticles is clearly shown in the inset in Figure 4a, which highlights also their tendency to
due to their spontaneous magnetic nature. Figure 4b shows the CA/5-LG/Cu-ferrite NCs, where the
aggregate in clusters, due to their spontaneous magnetic nature. Figure 4b shows the CA/5-LG/
formation of the NCs is evident, which are much larger than the NPs of bare Cu-ferrite illustrated in
Cu-ferrite NCs, where the formation of the NCs is evident, which are much larger than the NPs of bare
Figure 4a. It seems from Figure 4b that the CA polymer with the lemongrass essential oil has well
Cu-ferrite illustrated in Figure 4a. It seems from Figure 4b that the CA polymer with the lemongrass
covered the Cu-ferrite NPs in accordance with the AFM images shown before (Figure 3). Similarly, the
essential oil has well covered the Cu-ferrite NPs in accordance with the AFM images shown before
TEM image in Figure 4c shows the morphology of bare Cu-ferrite NPs that tend to aggregate due to
(Figure 3). Similarly, the TEM image in Figure 4c shows the morphology of bare Cu-ferrite NPs that
their spontaneous magnetic nature. In Figure 4d, the TEM image of CA/5-LG NCs is shown, where
tend to aggregate due to their spontaneous magnetic nature. In Figure 4d, the TEM image of CA/5-LG
it is clear that the NCs are spherical with a diameter up to 200 nm. In Figure 4e, the TEM image of
NCs is shown, where it is clear that the NCs are spherical with a diameter up to 200 nm. In Figure 4e,
CA/5-LF/Cu-ferrite NCs is illustrated, where a clear distinction between the NCs with and without
the TEM image of CA/5-LF/Cu-ferrite NCs is illustrated, where a clear distinction between the NCs
Cu-ferrite NPs is shown. In Figure 4e, the morphology of the NCs is not as spherical as before: they
with and without Cu-ferrite NPs is shown. In Figure 4e, the morphology of the NCs is not as spherical
tend to aggregate with each other; they have features that resemble tetragonal or cubic structures arising
as before: they tend to aggregate with each other; they have features that resemble tetragonal or cubic
upon assembly with Cu-ferrites; and they have a larger diameter, more than 200 nm. Energy dispersive
structures arising upon assembly with Cu-ferrites; and they have a larger diameter, more than 200 nm.
X-ray spectroscopy analysis (EDS) was used to confirm the Cu-ferrite nature on the CA/5-LG/Cu-ferrite
Energy dispersive X-ray spectroscopy analysis (EDS) was used to confirm the Cu-ferrite nature on
NCs, and it is shown in Supplementary Materials Figure S1. The chemical composition of the Cu-ferrites
the CA/5-LG/Cu-ferrite NCs, and it is shown in Supplementary Materials Figure S1. The chemical
on dried CA/5-LG/Cu-ferrite NCs was revealed with EDS as the Cu, Fe and O elements presented
composition of the Cu-ferrites on dried CA/5-LG/Cu-ferrite NCs was revealed with EDS as the Cu,
in the Cu-ferrite NPs (Figure S1). The % composition of the elements was O = 51.48%, Fe = 33.00%,
Fe and O elements presented in the Cu-ferrite NPs (Figure S1). The % composition of the elements was
Cu = 15.52%.
O = 51.48%, Fe = 33.00%, Cu = 15.52%.
In Figure 5, the HAADF-STEM and STEM-EDS data on a selected area of CA/5-LG/Cu-ferrite
In Figure 5, the HAADF-STEM and STEM-EDS data on a selected area of CA/5-LG/Cu-ferrite
NCs dried on a grid are shown. We can see that the Cu-ferrites are localized in some areas around
NCs dried on a grid are shown. We can see that the Cu-ferrites are localized in some areas around the
the polymeric capsules, and the SAED pattern proved their tetragonal structure. It was noticed that
polymeric capsules, and the SAED pattern proved their tetragonal structure. It was noticed that the
the combination of the vacuum inside the TEM together with the application of the electron beam
combination of the vacuum inside the TEM together with the application of the electron beam (200-kV
(200-kV acceleration voltage) was creating instability of the capsules and was decomposing the
acceleration voltage) was creating instability of the capsules and was decomposing the structure of
structure of the cellulose acetate. In the solution, it is concluded from the DLS data shown before that
the cellulose acetate. In the solution, it is concluded from the DLS data shown before that the NCs
the NCs preferentially remain as independent capsules without aggregating effects.
preferentially remain as independent capsules without aggregating effects.
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Figure 4. (a) SEM of Cu-ferrite NPs (in the inset, particles of ca. 70 nm are highlighted with red bars);
Figure
(a) of
SEM
of Cu-ferrite NPs
(in(c)the
inset,
particles NPs;
of ca.(d)70TEM
nm of
areCA/5-LG
highlighted
(b)4.SEM
CA/5-LG/Cu-ferrite
NCs;
TEM
of Cu-ferrite
NCs; with
and red
bars);(e)
(b)
SEM
of
CA/5-LG/Cu-ferrite
NCs;
(c)
TEM
of
Cu-ferrite
NPs;
(d)
TEM
of
CA/5-LG
NCs;
TEM4.of(a)
CA/5-LG/Cu-ferrite
Figure
SEM of Cu-ferrite NCs.
NPs (in the inset, particles of ca. 70 nm are highlighted with red bars);
and (e)
NCs.(c) TEM of Cu-ferrite NPs; (d) TEM of CA/5-LG NCs; and
(b)TEM
SEMofofCA/5-LG/Cu-ferrite
CA/5-LG/Cu-ferrite NCs;
(e) TEM of CA/5-LG/Cu-ferrite NCs.
Figure 5. (a) HAADF-STEM and (b–e) corresponding STEM-EDS maps of Fe, Cu, O and C distribution
over a region of the sample. The quantification of the (f) corresponding EDS spectrum gives atomic
wt
% Cu:Fe:O:C
= 0.5:0.98:1.0:97.52;
SAED pattern
from Cu-ferrite
andO(h)
Figure
5. (a) HAADF-STEM
and (b–e)(g)
corresponding
STEM-EDS
maps ofNPs
Fe, Cu,
andcorresponding
C distribution
Figure
5.
(a)
HAADF-STEM
and
(b–e)
corresponding
STEM-EDS
maps
of
Fe,
Cu,
O and
C distribution
azimuthal
integration,
compared
to
the
database
pattern
for
tetragonal
CuFe2O4
(ICSD
188855).
over a region of the sample. The quantification of the (f) corresponding EDS spectrum
gives
atomic
over awt
region
of the sample.
The quantification
the (f)from
corresponding
EDS
spectrum
gives atomic
% Cu:Fe:O:C
= 0.5:0.98:1.0:97.52;
(g) SAEDof
pattern
Cu-ferrite NPs
and
(h) corresponding
wt % azimuthal
Cu:Fe:O:Cintegration,
= 0.5:0.98:1.0:97.52;
pattern
Cu-ferrite
NPs and
(h)188855).
corresponding
compared to(g)
theSAED
database
patternfrom
for tetragonal
CuFe2O4
(ICSD
azimuthal integration, compared to the database pattern for tetragonal CuFe2O4 (ICSD 188855).
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2.5. Magnetic Measurements
2.5. Magnetic Measurements
Magnetization measurement was performed at 5 K and room temperature (300K) on bare and
Magnetization
measurement
was performed
at 5 K and
room temperature
on bare
and6a)
CA-encapsulated
Cu-ferrite
NPs (Figure
6). The magnetic
behavior
of the bare (300K)
Cu-ferrite
(Figure
CA-encapsulated
Cu-ferrite
NPs
(Figure
6).
The
magnetic
behavior
of
the
bare
Cu-ferrite
(Figure
6a)
is
is typical of a system of ferro-/ferri-magnetic nanoparticles in the blocked state. The temperature
typical
of
a
system
of
ferro-/ferri-magnetic
nanoparticles
in
the
blocked
state.
The
temperature
dependence of the magnetization can be understood on the basis of the thermal fluctuation of the
dependence
of while
the magnetization
be understood
on the
basis of thesuggests
thermal fluctuation
of the of
magnetic
spins,
the decreasecan
of the
coercivity with
temperature
the approaching
magnetic
spins,
while
the
decrease
of
the
coercivity
with
temperature
suggests
the
approaching
of
the nanoparticle systems to the superparamagnetic regime. The magnetization of saturation, reached
the
nanoparticle
systems
to
the
superparamagnetic
regime.
The
magnetization
of
saturation,
reached
for ca. 3 kOe, is consistent with literature data [21]. When Cu-ferrite nanoparticles are encapsulated
forCA
ca. 3with
kOe,and
is consistent
with
literature
data [21].
When
Cu-ferrite nanoparticles
are encapsulated
with
without LG
(sample
containing
LG,
CA/5-LG/Cu-ferrite
NCs, shown
in Figure 6b),
with CA with and without LG (sample containing LG, CA/5-LG/Cu-ferrite NCs, shown in Figure 6b),
the magnetic behavior of the composite is completely dominated by the diamagnetic contribution
the magnetic behavior of the composite is completely dominated by the diamagnetic contribution of
of the matrix, evidenced by the linear trend with the negative slope reported in Figure 6b. The very
the matrix, evidenced by the linear trend with the negative slope reported in Figure 6b. The very
weak magnetic contribution from Cu-ferrite nanoparticles can be still observed below 2 kOe magnetic
weak magnetic contribution from Cu-ferrite nanoparticles can be still observed below 2kOe magnetic
field intensities, as shown in the inset of the graph. The observed behavior is justified by the very
field intensities, as shown in the inset of the graph. The observed behavior is justified by the very low
low
concentration
Cu-ferrite
NPs
sample,
which,
from
a very
rough
estimation
obtained
concentration
of of
thethe
Cu-ferrite
NPs
in in
thethe
sample,
which,
from
a very
rough
estimation
obtained
comparing
the
as low
low as
as 0.1–0.2
0.1–0.2per
perthousand.
thousand.
comparing
themagnetization
magnetizationcurves
curvesof
ofFigure
Figure 5,
5, is
is as
Figure 6. Magnetization curve of (a) Cu-ferrite nanopowder and (b) lyophilized CA/5-LG/Cu-ferrite
Figure 6. Magnetization curve of (a) Cu-ferrite nanopowder and (b) lyophilized CA/5-LG/Cu-ferrite
NCs measured at 5 K and 300 K; the insets show a magnification of the corresponding hysteresis loop
NCs measured at 5 K and 300 K; the insets show a magnification of the corresponding hysteresis loop
in the low field range.
in the low field range.
We assume that the low response of mixed CA/5-LG/Cu-ferrite NCs to an external magnetic field
We
assume
the low
mixed
CA/5-LG/Cu-ferrite
to an external
magnetic
will reduce
the that
possibility
toresponse
use these of
NCs
to direct
them to a specific NCs
target-tissue
for biomedical
field
will reduce
possibility
to use magnetic
these NCsgradient
to direct
them to but
a specific
target-tissue
for biomedical
applications
bythe
means
of an external
[12,24,25],
these NCs
will be advantageous
in other applications
where
antimicrobial
drug
delivery[12,24,25],
is neededbut
without
such
applications
by means of
an external
magnetic
gradient
theseaggregating
NCs will beeffects,
advantageous
skinapplications
and mucosalwhere
infections
[8,26] or for
other
biomedical
applications
Furthermore,
thesuch
low as
in as
other
antimicrobial
drug
delivery
is needed
without[27].
aggregating
effects,
magnetic
properties
of
CA/5-LG/Cu-ferrite
NCs
are
advantageous,
since
Cu-ferrites
have
spontaneous
skin and mucosal infections [8,26] or for other biomedical applications [27]. Furthermore, the low
magnetism
and tend
to cluster together; whereas
clustering can
avoided when
polymeric
magnetic
properties
of CA/5-LG/Cu-ferrite
NCs aresuch
advantageous,
sincebeCu-ferrites
have spontaneous
NCs,
such
as
the
current
CA,
is
involved,
leaving
the
NCs
apart
and
available
for drug
magnetism and tend to cluster together; whereas such clustering can be avoided when polymeric
NCs,
delivery
applications.
such as the current CA, is involved, leaving the NCs apart and available for drug delivery applications.
Raman
Spectroscopy
2.6.2.6.
Raman
Spectroscopy
Ramanspectra
spectraofofCA/Cu-ferrite
CA/Cu-ferrite NCs,
NCs, CA/5-LG/Cu-ferrite
CA/5-LG/Cu-ferrite NCs
and
bare
LGLG
essential
oil oil
areare
Raman
NCs
and
bare
essential
−1, which are associated
presented
in
Figure
7.
LG
oil
has
two
characteristic
peaks
at
1625
and
1670
cm
´
1
presented in Figure 7. LG oil has two characteristic peaks at 1625 and 1670 cm , which are associated
with C=C and C=O bonds arising from the two aldehydes of LG; neral and geranial [2,28]. These two
with C=C and C=O bonds arising from the two aldehydes of LG; neral and geranial [2,28]. These
peaks due to the C=C and C=O bonds appeared also in the CA/5-LG/Cu-ferrite NCs, but shifted at
two peaks due to the C=C and −1C=O bonds appeared
also in the CA/5-LG/Cu-ferrite NCs, but shifted
higher wavelengths, 1655 cm and 1682 cm−1, respectively, due to hemi-acetal reactions [29–31] that
at higher wavelengths, 1655 cm´1 and 1682 cm´1 , respectively, due to hemi-acetal reactions [29–31]
take place between the aldehyde molecules in LG essential oil and the OH groups of the CA matrix.
that take place between the aldehyde molecules in LG essential oil and the OH groups of the CA matrix.
This hemi-acetal formation has been shown before for CA/5-LG NCs, but the C=C and C=O bonds
This
hemi-acetal
formation
haswavelengths
been showncompared
before fortoCA/5-LG
NCs, but the C=C
C=O bonds
appeared
at slightly
different
the CA/5-LG/Cu-ferrite
NCs; and
the reason
for
Molecules 2016, 21, 520
7 of 14
Molecules 2016, 21, 520
7 of 14
appeared at slightly different wavelengths compared to the CA/5-LG/Cu-ferrite NCs; the reason for
this
Cu-ferrites
that
change
thethe
absorbance
wavelength
of the
and
this shift
shift should
shouldbe
bethe
thepresence
presenceofof
Cu-ferrites
that
change
absorbance
wavelength
of C=C
the C=C
C=O
bonds.
and C=O
bonds.
Figure 7.
7. Raman
Raman spectra
spectra of
of LG
LG essential
essential oil
oil (blue
(blue dots),
dots),CA/Cu-ferrite
CA/Cu-ferrite NCs
(black dashed
dashed line)
line) and
and
Figure
NCs (black
CA/5-LG/Cu-ferrite NCs
CA/5-LG/Cu-ferrite
NCs(brown
(brownline).
line).
2.7. UV-VIS Absorption Spectroscopy
2.7. UV-VIS Absorption Spectroscopy
Before proceeding with the antimicrobial analysis, it was necessary to evaluate the amount of
Before proceeding with the antimicrobial analysis, it was necessary to evaluate the amount of LG
LG present in 50 μL of CA/5-LG/Cu-ferrite NCs solution used for the antimicrobial analysis. UV spectra
present in 50 µL of CA/5-LG/Cu-ferrite NCs solution used for the antimicrobial analysis. UV spectra
of 10 μL CA/5-LG/Cu-ferrite NCs and of 50 μL CA/5-LG NCs are shown in Figure 8a. The reason why
of 10 µL CA/5-LG/Cu-ferrite NCs and of 50 µL CA/5-LG NCs are shown in Figure 8a. The reason
we used 10 μL CA/5-LG/Cu-ferrite NCs was that the 50 μL CA/5-LG/Cu-ferrite NCs saturate the UV
why we used 10 µL CA/5-LG/Cu-ferrite NCs was that the 50 µL CA/5-LG/Cu-ferrite NCs saturate
absorption signal. Moreover, UV spectra of bare LG essential oil were acquired with increasing amounts
the UV absorption signal. Moreover, UV spectra of bare LG essential oil were acquired with increasing
of LG to obtain a calibration curve of the intensity of the peaks at 238 nm vs. the LG volume, as shown
amounts of LG to obtain a calibration curve of the intensity of the peaks at 238 nm vs. the LG volume,
in Figure 8b. To calculate the amount of LG in 10 μL CA/5-LG/Cu-ferrite NCs and of 50 μL of CA/5-LG
as shown in Figure 8b. To calculate the amount of LG in 10 µL CA/5-LG/Cu-ferrite NCs and of 50 µL
NC solution, the intensity of the UV spectrum at the 240-nm peak was extrapolated on the calibration
of CA/5-LG NC solution, the intensity of the UV spectrum at the 240-nm peak was extrapolated on the
curve. UV analysis on 10 μL of CA/5-LG/Cu-ferrite NCs (Figure 7) showed that the content of LG
calibration curve. UV analysis on 10 µL of CA/5-LG/Cu-ferrite NCs (Figure 7) showed that the content
inside the capsules was about 1.2 μL. Therefore, in 50 μL of the CA/5-LG/Cu-ferrite NCs that used
of LG inside the capsules was about 1.2 µL. Therefore, in 50 µL of the CA/5-LG/Cu-ferrite NCs that
for the antimicrobial experiment, as will be shown below, there were 6 μL or 5.322 μg of LG, which
used for the antimicrobial experiment, as will be shown below, there were 6 µL or 5.322 µg of LG, which
was higher than the amount of LG found inside the CA/5-LG NCs without Cu-ferrites published
was higher than the amount of LG found inside the CA/5-LG NCs without Cu-ferrites published
before (0.9 μL), even if the initial concentration of LG used to prepare the NCs was the same [8]. This
before (0.9 µL), even if the initial concentration of LG used to prepare the NCs was the same [8]. This
showed that the presence of Cu-ferrite NPs enhanced the attachment and, hence, the concentration
showed that the presence of Cu-ferrite NPs enhanced the attachment and, hence, the concentration and
and stabilization of LG inside the CA/5-LG/Cu-ferrite NCs, a result that is in accordance with other
stabilization of LG inside the CA/5-LG/Cu-ferrite NCs, a result that is in accordance with other work
work on different essential oils, vanilla, patchouli and ylang ylang [32], as well as Satureja hortensis
on different essential oils, vanilla, patchouli and ylang ylang [32], as well as Satureja hortensis [33]
[33] stabilized into iron oxide nanoparticles, with antimicrobial properties. To exclude the
stabilized into iron oxide nanoparticles, with antimicrobial properties. To exclude the interference
of
interference of Cu-ferrite and CA absorption in the wavelength of around
238–240 cm−1, UV spectra
´
1
Cu-ferrite and CA absorption in the wavelength of around 238–240 cm , UV spectra were acquired
were acquired and are shown in Figure S2.
and are shown in Figure S2.
A potential reaction that occurs between Cu-ferrite NPs and CA polymer is with the reaction of
A potential reaction that occurs between Cu-ferrite NPs and CA polymer is with the reaction
oxygen-rich groups, such as acetate groups or OH groups in the CA molecule, with Cu-ferrite. Similar
of oxygen-rich groups, such as acetate groups or OH groups in the CA molecule, with Cu-ferrite.
reaction schemes between CA and iron [34] or copper [35] NPs have been shown before, where the
Similar reaction schemes between CA and iron [34] or copper [35] NPs have been shown before, where
O-rich groups of CA react with the metallic NPs. Taking into account also the mentioned UV studies,
the O-rich groups of CA react with the metallic NPs. Taking into account also the mentioned UV
where a clear enhancement of the LG oil was present in the CA/5-LG/Cu-ferrite NCs, we also propose
studies, where a clear enhancement of the LG oil was present in the CA/5-LG/Cu-ferrite NCs, we
that some of the LG oil molecules, such as the prevailing aldehydes, react with the Cu-ferrite NPs,
also propose that some of the LG oil molecules, such as the prevailing aldehydes, react with the
creating micelles with steric hindrance on them and not allowing the metallic NPs to aggregate. Such
Cu-ferrite NPs, creating micelles with steric hindrance on them and not allowing the metallic NPs to
electro-steric stabilization has been also shown previously on Au NPs with carboxylic acids of
Anacardium occidentale essential oils [36]. Therefore, a proposed mechanism of the NC’s formation
is presented in Figure 9, where aldehyde molecules from essential oils can be bound to both OH groups
Molecules 2016, 21, 520
8 of 14
Molecules
2016,
21, 520
aggregate.
Such
electro-steric
8 of 14
stabilization has been also shown previously on Au NPs with carboxylic
acids of Anacardium occidentale essential oils [36]. Therefore, a proposed mechanism of the NC’s
of
CA Molecules
creating
hemi-acetal
bonds 9,
[8]where
and onaldehyde
metallic molecules
NPs electrostatically.
It is oils
alsocan
mentioned
2016,
21, 520 in Figure
8 of 14here
formation
is presented
from essential
be bound
to
that
other
polar
molecules
from
LG
essential
oil,
such
as
alcohols
and
acids,
can
also
be
bound
to
both OH groups of CA creating hemi-acetal bonds [8] and on metallic NPs electrostatically. It is also
of CA
creating hemi-acetal bonds
[8] and 9,
onthe
metallic NPs electrostatically.
It is alsoNPs
mentioned
here
Cu-ferrite
similarly.
in Figure
of metallic
Cu-ferrite
withacids,
the
acetyl
mentioned
here thatFurthermore,
other polar molecules
fromformation
LG essential
oil, such
as alcohols
and
can
that other polar molecules from LG essential oil, such as alcohols and acids, can also be bound to
or
hydroxyl
groups
of
CA
is
shown.
To
prove
the
existence
of
LG
oil
in
both
CA/5-LG
NCs
and
CA/
also beCu-ferrite
bound to
Cu-ferrite
similarly.inFurthermore,
in Figure
the formation
metallic
similarly.
Furthermore,
Figure 9, the formation
of 9,
metallic
Cu-ferrite of
NPs
with theCu-ferrite
acetyl
5-LG/Cu-ferrite
NCs as
bound
to the
CA polymer
and/or
Cu-ferrite
NPs,
thermogravimetric
NPs with
the
acetyl
or
hydroxyl
groups
of
CA
is
shown.
To
prove
the
existence
of
LGand
oilanalysis
in both
or hydroxyl groups of CA is shown. To prove the existence of LG oil in both CA/5-LG NCs
CA/
(TGA)
on
lyophilized
samples
was
performed
as
shown
in
Supplementary
Materials
Figures
S3–S5,
CA/5-LG
NCs and CA/5-LG/Cu-ferrite
as bound
the CANPs,
polymer
and/or Cu-ferrite
NPs,
5-LG/Cu-ferrite
NCs as bound to the CANCs
polymer
and/or to
Cu-ferrite
thermogravimetric
analysis
for
samples
with
and
without
LG
oil.
As
you
can
see,
the
weight
loss
of
CA
NPs
without
LG
oil is
(TGA) on lyophilized
performed as
shown in
Supplementary
Figures
S3–S5,
thermogravimetric
analysissamples
(TGA) was
on lyophilized
samples
was
performed asMaterials
shown in
Supplementary
very
different
from
theand
NCs
with LG
LGwith
oil,As
where
the see,
lastLG
ones
started
loss
their
weight
earlier
for samples
with
without
oil.
youwithout
can
the
weight
losstoof
CA
NPs
without
oil of
isdue
Materials
Figures
S3–S5,
for
samples
and
oil.
As you
can
see,
the
weightLG
loss
CA
very
different
from
the
NCs
with
LG
oil,
where
the
last
ones
started
to
loss
their
weight
earlier
due
to
the
high
volatility
of
LG
oil
at
lower
temperatures
compared
to
the
CA
polymer.
NPs without LG oil is very different from the NCs with LG oil, where the last ones started to loss their
the high volatility of LG oil at lower temperatures compared to the CA polymer.
weighttoearlier
due to the high volatility of LG oil at lower temperatures compared to the CA polymer.
8. (a) UV-VIS
absorption
spectra
of the
10-μL
CA/5-LG/Cu-ferrite NC
CA/5-LG
NC NC
FigureFigure
8. (a) UV-VIS
absorption
spectra
of the
10-µL
CA/5-LG/Cu-ferrite
NCand
and50-μL
50-µL
CA/5-LG
Figuresolution
8. (a) UV-VIS
absorption
spectra
of
thecalculation
10-μL CA/5-LG/Cu-ferrite
NC
and
50-μL CA/5-LG NC
and
(b)
the
calibration
curve
and
of
LG
oil
content
in
10
μL
CA/5-LG/Cu-ferrite
solution and (b) the calibration curve and calculation of LG oil content in 10 µL CA/5-LG/Cu-ferrite
solution
and μL
(b)CA/5-LG
the calibration
NCs. curve and calculation of LG oil content in 10 μL CA/5-LG/Cu-ferrite
and 50and
µL 50
CA/5-LG
NCs.
and 50 μL CA/5-LG NCs.
Figure 9. Molecular diagram of the reactions that can take place between aldehyde molecules,
Cu-ferrite NPs and the hydroxyl group of the cellulose acetate ring to produce electrostatic bonds and
hemiacetal bonds, respectively. The reactions between the Cu-ferrite NP with the acetyl group (R)
Figure 9. Molecular diagram of the reactions that can take place between aldehyde molecules,
Figureand/or
9. Molecular
diagram
of the
that can
place between
aldehyde molecules, Cu-ferrite
3.
OH group
of CA are
alsoreactions
visible, where
R istake
OC(=O)CH
Cu-ferrite NPs and the hydroxyl group of the cellulose acetate ring to produce electrostatic bonds and
NPs and the hydroxyl group of the cellulose acetate ring to produce electrostatic bonds and hemiacetal
hemiacetal
bonds,
respectively.
The reactions
between the Cu-ferrite NP with the acetyl group (R)
2.8. Inductively
Coupled
Mass
Spectrometry
bonds,
respectively.
ThePlasma
reactions
between
the Cu-ferrite NP with the acetyl group (R) and/or OH
and/or OH group of CA are also visible, where R is OC(=O)CH3.
group ICP
of CA
are also visible,
where R is OC(=O)CH
3 . solution calculated the concentration of Cu and
measurements
of CA/5-LG/Cu-ferrite
NC
Fe, which was found to be 21.75 ppm and 28.3 ppm, respectively. The amounts of Cu and Fe found
2.8. Inductively Coupled Plasma Mass Spectrometry
in 50 μL of
CA/5-LG/Cu-ferrite
solution used for the antimicrobial experiments were therefore
2.8. Inductively
Coupled
Plasma MassNC
Spectrometry
1.0875
and 1.415 μg, of
respectively;
taking into account
that ppm
is expressed
mg/L.
ICP
measurements
CA/5-LG/Cu-ferrite
NC solution
calculated
theas
concentration
of Cu and
ICP measurements of CA/5-LG/Cu-ferrite NC solution calculated the concentration of Cu and
Fe, which was found to be 21.75 ppm and 28.3 ppm, respectively. The amounts of Cu and Fe found
Fe, which
was found to
be 21.75 ppm and 28.3 ppm, respectively. The amounts of Cu and Fe found
2.9. Antimicrobial
Tests
in 50 μL
of CA/5-LG/Cu-ferrite
NC solution used for the antimicrobial experiments were therefore
in 50 µL of CA/5-LG/Cu-ferrite NC solution used for the antimicrobial experiments were therefore
formed
NCs were tested
against
aureus bacteria.
We
thatas
bare
CA NPs had no
1.0875 andThe
1.415
μg, respectively;
taking
intoS.
account
that ppm
is observe
expressed
mg/L.
1.0875effect
and 1.415
µg, respectively;
taking
that ppm
expressed
as Both
mg/L.
on bacteria,
as expected
frominto
the account
inert nature
of theis CA
polymer.
CA/5-LG and
2.9. Antimicrobial Tests
The formed NCs were tested against S. aureus bacteria. We observe that bare CA NPs had no
effect on bacteria, as expected from the inert nature of the CA polymer. Both CA/5-LG and
Molecules
Molecules 2016,
2016, 21,
21, 520
520
2.9.
2.9. Antimicrobial
Antimicrobial Tests
Tests
Molecules 2016, 21, 520
99 of
of 14
14
9 of 14
The
The formed
formed NCs
NCs were
were tested
tested against
against S.
S. aureus
aureus bacteria.
bacteria. We
We observe
observe that
that bare
bare CA
CA NPs
NPs had
had
CA/Cu-ferrite
NCs
were
found
to
have
very
good
antimicrobial
activities
when
encapsulated
intoand
the
no
no effect
effect on
on bacteria,
bacteria, as
as expected
expected from
from the
the inert
inert nature
nature of
of the
the CA
CA polymer.
polymer. Both
Both CA/5-LG
CA/5-LG
and
CA
polymer. In
particular,
theto
of
Cu-ferrite
was betteractivities
than that
of LG,
as illustrated
in
CA/Cu-ferrite
NCs
were
have
good
when
encapsulated
into
CA/Cu-ferrite
NCs
were found
found
toeffect
have very
very
good antimicrobial
antimicrobial
activities
when
encapsulated
into the
the
Figure
10,
with
about
50%
more
efficacy.
However,
the
combination
of
both
LG
and
Cu-ferrite
led
to
CA
polymer.
In
particular,
the
effect
of
Cu-ferrite
was
better
than
that
LG,
as
illustrated
in
Figure
10,
CA polymer. In particular, the effect of Cu-ferrite was better than that of LG, as illustrated in Figure 10,
very
efficient
as the
couldof
even
after 20led
hours
of efficient
culture
with
50%
more
However,
the
combination
both
LG
Cu-ferrite
to
with about
about
50%antimicrobial
more efficacy.
efficacy.NCs,
However,
thebacteria
combination
ofnot
bothgrow
LG and
and
Cu-ferrite
led
to very
very
efficient
(Figure
10).
This
is
a
very
important
result,
since
it
shows
that
a
combination
of
organic
and
inorganic
antimicrobial
NCs,
as
the
bacteria
could
not
grow
even
after
20
hours
of
culture
(Figure
10).
antimicrobial NCs, as the bacteria could not grow even after 20 hours of culture (Figure 10). This
This is
is
antimicrobial
substances
in
a
polymeric
matrix
can
produce
a
nanomaterial
with
extremely
good
aa very
important
result,
since
it
shows
that
a
combination
of
organic
and
inorganic
antimicrobial
very important result, since it shows that a combination of organic and inorganic antimicrobial
antimicrobial
is produce
believed aatonanomaterial
have two positive
effects ongood
the antimicrobial
substances
aa polymeric
matrix
with
substances in
in properties.
polymericCu-ferrite
matrix can
can
produce
nanomaterial
with extremely
extremely
good
antimicrobial
activity;
to
provide
an
additional
antimicrobial
substance
to
the
NCs
and
to
stabilize
and
increase
the
properties.
Cu-ferrite
is
believed
to
have
two
positive
effects
on
the
antimicrobial
activity;
to
properties. Cu-ferrite is believed to have two positive effects on the antimicrobial activity;
to provide
provide
LG
content
inside
the
NCs.
Cu-ferrite
NPs
are
supposed
to
adhere
to
bacterial
cell
wall
and
penetrate
an
an additional
additional antimicrobial
antimicrobial substance
substance to
to the
the NCs
NCs and
and to
to stabilize
stabilize and
and increase
increase the
the LG
LG content
content inside
inside
through
the
cell
membrane;
while
copper
ions
cause
destruction
of
the
bacterial
cell
wall,
degradation
the
NCs.
Cu-ferrite
NPs
are
supposed
to
adhere
to
bacterial
cell
wall
and
penetrate
through
the
the NCs. Cu-ferrite NPs are supposed to adhere to bacterial cell wall and penetrate through the cell
cell
and
lysis of the
cytoplasm
and, cause
therefore,
cell death
Therefore,
the
excellent antimicrobial
membrane;
while
copper
destruction
of
the
cell
degradation
and
membrane;
while
copper ions
ions
cause
destruction
of [16,37,38].
the bacterial
bacterial
cell wall,
wall,
degradation
and lysis
lysis of
of
efficacy
of the CA/5-LG/Cu-ferrite
NCs was
due to two
reasons:
first
that theantimicrobial
presence of Cu-ferrites
the
and,
[16,37,38].
Therefore,
the
excellent
efficacy
the cytoplasm
cytoplasm
and, therefore,
therefore, cell
cell death
death
[16,37,38].
Therefore,
the
excellent
antimicrobial
efficacy of
of
led
an enhancement ofNCs
LG essential
oiltwo
concentration,
asthat
shown
by
UV studies,
and second,
the
CA/5-LG/Cu-ferrite
was
reasons:
the
of
led
an
the to
CA/5-LG/Cu-ferrite
NCs
was due
due to
to
two
reasons: first
first
that
the presence
presence
of Cu-ferrites
Cu-ferrites
led to
tothe
an
presence
of
Cu-ferrite
inside
the
NCs
change
their
morphology
and
chemistry,
and
thus,
presence
enhancement
of
LG
essential
oil
concentration,
as
shown
by
UV
studies,
and
second,
the
enhancement of LG essential oil concentration, as shown by UV studies, and second, the presence
of
both
antimicrobial
LG essential
oil and Cu-ferrites
in the NCs
bacterial
cell
inside
NCs
their
and
and
thus,
presence
of
of Cu-ferrite
Cu-ferrite
inside the
theagents
NCs change
change
their morphology
morphology
and chemistry,
chemistry,
and increase
thus, the
thethe
presence
of both
both
death.
antimicrobial
agents
LG
essential
oil
and
Cu-ferrites
in
the
NCs
increase
the
bacterial
cell
death.
antimicrobial agents LG essential oil and Cu-ferrites in the NCs increase the bacterial cell death.
Figure
10.
Antimicrobial
activity
of
CA/5-LG,
CA/Cu-ferrite
and CA/5-LG/Cu-ferrite
CA/5-LG/Cu-ferriteNCs.
NCs.
Figure10.
10.Antimicrobial
Antimicrobialactivity
activityof
ofCA/5-LG,
CA/5-LG, CA/Cu-ferrite
CA/Cu-ferrite and
Figure
and
CA/5-LG/Cu-ferrite
NCs.
3.
3. Experimental
ExperimentalSection
Section
3.
Experimental
Section
3.1. Materials
Materials
3.1.
Materials
LG essential
essential oil
oil (100%
(100% pure)
pure) was
was purchased
purchased from
from Maitreya-Natura
Maitreya-Natura (Pesaro-Urbino,
(Pesaro-Urbino, Italy).
Italy).
LG
essential
Maitreya-Natura
(Pesaro-Urbino,
Italy).
Cellulose
acetate
(CA)
(acetyl
content
of
39.8%;
MW
1/4
30
kDa)
and
acetone
were
purchased
Cellulose
39.8%;
MW
1/4 1/4
30 kDa)
and acetone
were were
purchased
from
Cellulose acetate
acetate(CA)
(CA)(acetyl
(acetylcontent
contentofof
39.8%;
MW
30 kDa)
and acetone
purchased
from Sigma
Sigma
Aldrich,
Milano,
Italy.
Milli-Q
water was
was used
used for
formethod
the solvent/antisolvent
solvent/antisolvent
method
Sigma
Aldrich.
Milli-QMilano,
water was
usedMilli-Q
for the water
solvent/antisolvent
for the precipitation
of the
from
Aldrich,
Italy.
the
method
for the
the precipitation
precipitation
of the
the nanoparticles.
nanoparticles.
Staphylococcus
aureus (S.
(S. aureus)
aureus)
was
purchased from
from
ATCC
nanoparticles.
Staphylococcus
aureus (S. aureus)
was purchased
from
ATCC
(Washington,
DC, USA).
for
of
Staphylococcus
aureus
was
purchased
ATCC
(Washington,
DC,
USA).
S.
aureus
was
grown
on
Luria-Bertani
(LB)
medium,
composed
of:
1%
tryptone
S.
aureus was grown
on Luria–Bertani
(LB) medium,
composed
of: medium,
1% tryptone
(Sigma of:
Aldrich,
Italy),
(Washington,
DC, USA).
S. aureus was grown
on Luria-Bertani
(LB)
composed
1% tryptone
(Sigma
Aldrich),
0.5%
yeast
extractand
(Sigma
Aldrich)
and 1%
1% NaCl
NaCl (Sigma
(Sigma Aldrich).
Aldrich).
0.5%
yeast
extract0.5%
(Sigma
Aldrich)
1% NaCl
(Sigma
Aldrich).
(Sigma
Aldrich),
yeast
extract
(Sigma
Aldrich)
and
3.2. Cellulose Acetate/Lemongrass Oil/Cu-Ferrite Nanocapsules Preparation
Cu-ferrite nanoparticles were synthesized by the citrate-gel auto-combustion technique using the
source materials ferric nitrate (Fe(NO3)3·9H2O) and copper nitrate (Cu(NO3)2·3H2O). The metal nitrates
were dissolved in 100 mL of distilled water and stirred for 15 min, and then, citric acid was added in
the ratio of 1:1 to the metal nitrate solution. Ammonia droplets of a 33% concentration were added to
adjust the pH of the mixture to 7. The solution was continuously stirred at 130 °C and then was
Molecules 2016, 21, 520
10 of 14
3.2. Cellulose Acetate/Lemongrass Oil/Cu-Ferrite Nanocapsules Preparation
Cu-ferrite nanoparticles were synthesized by the citrate-gel auto-combustion technique using
the source materials ferric nitrate (Fe(NO3 )3 ¨ 9H2 O) and copper nitrate (Cu(NO3 )2 ¨ 3H2 O). The metal
nitrates were dissolved in 100 mL of distilled water and stirred for 15 min, and then, citric acid was
added in the ratio of 1:1 to the metal nitrate solution. Ammonia droplets of a 33% concentration were
added to adjust the pH of the mixture to 7. The solution was continuously stirred at 130 ˝ C and then
was transferred into xerogel. The dried gel burns in self-propagating combustion to form the ferrite
nanostructure. The reaction causes a rapid release of gasses with a great mass loss leading to ferrite
nano-powder formation. The Cu-ferrite nano-powder was then collected, and 0.1 g of it was inserted
into 200 mL of Milli-Q water, which was placed in an ultrasonic bath (1 min at 40 KHz) prior to its use.
Then, 100 mL of CA (10% m/v)/LG essential oil (5% v/v) was inserted drop-wise into the aqueous
Cu-ferrite solution. This mechanism of the formation of NCs is described in a previous article [8],
where CA and LG are chemically bonded, forming hemi-acetal bonds between the hydroxyl groups of
the cellulose acetate and the aldehyde groups of LG essential oil. Cu-ferrite is supposed to be formed
with the NCs by the aid of the hydrocarbon tails found in LG essential oil. Then, the precipitated
NCs were filtered using Sartorius 389F filters and left for 4 h under nitrogen flow until the remaining
acetone was evaporated. The number 5 before LG in CA/5-LG/Cu-ferrite NCs denotes the initial
volume percentage of LG essential oil.
3.3. Dynamic Light Scattering
Dynamic light scattering (DLS) measurements were acquired using a Zetasizer Nanoseries from
Malvern Instruments. The Z-average size was used in DLS, is a parameter also known as the cumulants
mean, defined as the “harmonic intensity averaged particle diameter” and is hydrodynamic in nature,
applicable to particles in dispersion.
3.4. X-ray Diffraction
X-ray diffraction (XRD) patterns were recorded on a Rigaku SmartLab X-Ray diffractometer
equipped with a 9-kW CuKα rotating anode (operating at 40 kV and 150 mA) and D\teX Ultra 1D
detector set in X-ray reduction mode. The diffraction patterns were collected at room temperature in
Bragg–Brentano geometry over an angular range 2θ from 15˝ to 85˝ , scan speed = 0.1˝ /min and step
size = 0.02˝ . XRD data analysis was carried out using PDXL 2.1 software from Rigaku.
3.5. Atomic Force Microscopy
For atomic force microscopy (AFM) measurements, a Park Systems AFM instrument (XE-100,
Suwon, Korea) was used in true non-contact mode. The images were acquired in air on an anti-vibration
table (Table Stable TS-150) and within an acoustic enclosure. Single-beam silicon cantilever tips
(PPP-NCHR-10) were used for the data acquisition with about less than a 10-nm nominal radius and
a 42-N/m elastic force constant for high sensitivity. The resonance frequency was defined around
280 kHz. The scan rate was maintained at 0.1 Hz.
3.6. High Resolution Scanning Electron Microscopy, Energy Dispersive X-ray Spectroscopy and Transmission
Electron Microscopy
High-resolution scanning electron microscopy (HRSEM) imaging was carried out using a JEOL
JSM 7500FA (Jeol, Tokyo, Japan) equipped with a cold field emission gun (cold-FEG), operating at a
15-kV acceleration voltage. Compositional contrast was achieved using a retractable backscattered
electron detector (RBEI), to better reveal the presence of Cu-ferrite within the CA-LG particle matrix.
Energy dispersive X-ray spectroscopy (EDS) was performed using an Oxford X-Max 80 system with
a silicon drift detector (SDD) having an 80-mm2 effective area of the detecting device. Samples for
HRSEM were prepared by drop casting 5 µL of solution onto a silicon wafer. Transmission electron
Molecules 2016, 21, 520
11 of 14
microscopy (TEM) images have been acquired with a JEOL JEM-1011 microscope, equipped with a
tungsten thermionic electron source and operated at 100 kV of acceleration voltage. Samples were
prepared by drop casting 20 µL of the solution onto carbon-coated copper grids. Selected-area electron
diffraction (SAED) patterns and high-angle annular dark field-scanning TEM images (HAADF-STEM)
images were acquired on a JEOL JEM-2200FS TEM (Schottky emitter, operated at 200 kV), equipped
with a Bruker Quantax400 systems and a XFlash 5060 SDD detector for STEM-EDS analyses. For EDS
analyses, the samples were deposited onto a C-coated Ni grid, and a holder without Cu parts was
used, so as to allow for Cu quantification.
3.7. Magnetic Measurements
The magnetic properties of the bare and encapsulated Cu-ferrite nanoparticles, with and without
LG, were investigated using a QuantumDesignMPMS SQUID magnetometer (QuantumDesign,
San Diego, CA, USA) operating in the temperature range 2 K/300 K with an applied field up to 5 Tesla.
Measurements were performed on the sample in solution and on lyophilized powder. The magnetic
moment was measured as a function of the field cycling between +5 T and ´5 T at 5 K and 300 K (room
temperature). The recorded magnetic moments were normalized for the total mass of the sample.
3.8. Raman Spectroscopy
A Horiba Jobin-Yvon µRaman operating with a He-Ne laser source was used to study the
molecular vibrations modes of bare LG oil, CA NPs and CA/5-LG/Cu-ferrite NCs. The wavelength
of the laser radiation was 632.8 nm, and the objective used was a 100ˆ with a slit aperture of about
200 µm.
3.9. UV-VIS Absorption Spectroscopy
A UV-VIS-NIR spectrophotometer by Varian (Cary 6000i) in double beam configuration was
used to study the LG oil content in the CA/5-LG/Cu-ferrite NCs. First, a calibration curve was
constructed by using bare LG essential oil in acetonitrile where the amount of the oil varied from
0.18 µL to 3.50 µL, as described analytically in a previous work [8]. To determine the amount of LG oil
in CA/5-LG/Cu-ferrite NC solution, the measured intensity values at 240 nm were extrapolated to the
calibration curve of bare LG. All of the UV measurements were done in acetonitrile solvent (3 mL).
3.10. Inductively Coupled Plasma Mass Spectrometry
To obtain the concentrations of Cu and Fe in the NCs, an ICP-OES spectrometer (iCAP 6300 DUO,
ThermoFisher) was used. A volume (400 µL) of NCs was digested in 10% of aqua regia (HCl/HNO3
3:1 (v/v)), incubated overnight at room temperature. Afterwards, Milli-Q water was added up to
10 mL and filtered first with an RC 0.45-µm filter to remove possible polymers in excess and lastly with
a PTFE 0.45 filter. After calibration of the instrument, the most sensitive wavelength for Cu (224.7 nm)
and Fe (238.2 nm) were considered.
3.11. Antimicrobial Tests
The biological properties of the produced NCs were tested against S. aureus (Gram-positive)
bacteria. Sterile conditions were used, and the NCs were manipulated inside a Class II biohazard
hood. For the antibacterial assays, cultures of S. aureus were grown overnight in LB medium in a
37 ˝ C incubator. The cultures were then diluted to obtain 106 CFUs/mL bacteria suspensions that
were placed in sterile 96 well plates, 50 µL in each well. Eight replicates were produced for the
tests. Immediately before starting the incubation, 50 µL of the NCs’ solution was added in each
well. The bacterial growth was recorded measuring the absorbance at 600 nm every 10 min by using
the microplate reader Fluo Star Optima (BMG LABTECH). The plates were maintained in culture
conditions for the duration of the experiment (37 ˝ C and double orbital shaking). Data were collected
Molecules 2016, 21, 520
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and elaborated with MARS DataAnalysis Sofware (BMG LABTECH). The negative control was then
the LB medium, whereas for the positive control, we used 10% DMSO.
4. Conclusions
We showed that Cu-ferrite NPs were immobilized in CA/5-LG NCs, increasing the size of
the final construct, CA/5-LG/Cu-ferrite NCs, to about 220–300 nm (from 150 nm of CA/5-LG
NCs). AFM, SEM and TEM studies showed that Cu-ferrite presence in the CA/5-LG/Cu-ferrite
NCs significantly changes the morphology of the NCs, making them larger, tetragonal and/or
cubic as opposed to spherical and smaller in the absence of Cu-ferrite, i.e., CA/5-LG NCs. The
CA/5-LG/Cu-ferrite NCs did not aggregate into the solution due to their immobilization in the CA
polymeric nanocapsules and their consequent magnetic hindrance, as found during the magnetic
studies that revealed that CA/5-LG/Cu-ferrite NCs have very weak magnetic properties, which
is advantageous for drug delivery applications when clustering effects due to the spontaneous
magnetization of Cu-ferrites should be avoided. The antimicrobial tests showed that CA/5-LG
and CA/Cu-ferrites have very good antimicrobial properties, but when both LG and Cu-ferrites
were used simultaneously (CA/5-LG/Cu-ferrite NCs), the antimicrobial effect enhances significantly.
The presence of LG oil is important for the stability of the NCs, since it also acts as a surfactant due to
its polar groups and hydrophobic chains. Indeed, Cu-ferrite alone with CA produces NCs that tend to
precipitate in a few hours, whereas the presence of LG produces stable NCs in water. The combination
of active agents, LG essential oil and Cu-ferrites in the CA polymer produces stable NCs and a higher
presence of LG essential oil in CA matrix compared to CA/5-LG NCs, as proven through the UV
analysis. Cu-ferrite is believed to be attached to some of the oxygen-rich groups of CA, such as acetyl
and hydroxyl groups, whereas LG essential oil was grafted at OH groups of CA with hemi-acetal
formation and on the Cu-ferrite with electrostatic interactions. This work opens novel routes on the
creation of NCs and NPs with the combination of organic and inorganic materials. The enhanced
antimicrobial property obtained by the combination of the two active organic/inorganic components
was tested on the S. aureus bacteria culture, the growth of which was reduced to zero, even after 20 h
of incubation with CA/5-LG/Cu-ferrite NCs.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/4/520/s1.
Acknowledgments: The authors would like to thank Athanassia Athanassiou for her useful discussion for the
preparation of this work and Paolo Di Fruscia for his kind help with the lyophilization of the nanocapsules.
Author Contributions: I.L.L. conceived and designed the experiments; M.H.A. prepared the Cu-Ferrite NPs; R.B.
and A.C. did the TEM, SAED and SEM analyis; C.I. did the magnetization studies; S.M. did the XRD analysis; R.C.
did the Raman spectroscopy; F.D. did the ICP analysis; V.B. and P.P. did the antimicrobial analysis; I.L.L. prepared
the CA/5-LG NPs and CA/5-LG/Cu-ferrite NCs, did the AFM, DLS and TGA experiments and wrote the paper,
with the contribution form the other authors on their parts.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds Cu-ferrite NPs, CA/5-LG NCs, CA/5-LG/Cu-ferrite NCs are
available from the authors.
© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).