Materials Science and Engineering C 57 (2015) 49–57

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Materials Science and Engineering C

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

Synthesis and functionalization of silica-based nanoparticles with

fluorescent biocompounds extracted from Eysenhardtia polystachya for
biological applications
Guadalupe Ferreira a, Angel R. Hernandez-Martinez a, Hector Pool b, Gustavo Molina a, Martha Cruz-Soto c,
Gabriel Luna-Barcenas b, Miriam Estevez a,⁎
a
Centro de Física Aplicada y Tecnología Avanzada (CFATA), Universidad Nacional Autónoma de México, Campus Juriquilla, Blvd. Juriquilla 3000, Juriquilla, Querétaro, Mexico
b
Centro de Investigación y Estudios Avanzados del IPN (CINVESTAV), Unidad Querétaro, Libramiento Norponiente #2000, Real de Juriquilla, C.P. 76230 Querétaro, Mexico
c
Universidad del Valle de México, Campus Querétaro, Blvd. Juriquilla 3000, Juriquilla, Querétaro, Mexico

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

Article history: Several types of dyes or fluorophores are used for the detection of interactions between drug carriers and cells,
Received 11 March 2015 within biomedicine field. However, many of them have a certain level of toxicity and instability affecting their bi-
Received in revised form 19 April 2015 ological properties. Different studies have demonstrated that nanoparticles (NPs) have interesting properties
Accepted 9 July 2015
that could be used to stabilize diverse biomolecules, including dyes. Here, we report the synthesis of a novel
Available online 14 July 2015
nanosystem by the functionalization of silica NPs using biocompounds extracted from Mexican tree “Palo azul”
Keywords:
(Eysenhardtia polystachya) and APTES as a coupling agent. Particle size, electrical properties, and morphology
Fluorescent isoflavones of the novel nanosystem were analyzed. The extracted biocompounds presented fluorescence which prevails
Silica nanoparticles over time, even after nanosystem formation and apparent cellular internalization. These were detected using
Biological applications MCF-7 cells visualized by confocal laser-scanning microscopy (CLSM), finding that the nanosystem was able to
Antiproliferative effects internalize into cells and act as a fluorescent biomarker.
MCF-7 cells By this method, our novel nanosystem opens the possibilities to obtain sensitive data in a noninvasive manner for
biological applications, such as early-stage cancer diagnosis, drug delivery, and pathogen detection.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
In recent years, the interest in formulation, study and application of
nanoparticles (NPs) in medicine, pharmaceutics, cosmetics and food
fields has been increased, mainly by their excellent properties as drug
delivery vectors as well as bioimaging and biosensing systems [1,2].
On the other hand, in the biomedicine field, the detection — in vitro
or/and in vivo — of interactions between drug carriers and cells is very
important for basic medical studies, and therapeutic interactions [3,4].
Different types of dyes or fluorophores are used for these studies;
however, many of them have a certain level of toxicity and instability
at different conditions (pH, temperature, among others), which hinders
their application in vivo [5,6]. Several studies, have demonstrated that
the physicochemical properties of different types of nanomaterials,
provide a higher dye stability [7,8] and sensitive detection from single-
molecule level to human body applications, i.e. from in vitro diagnosis to
in vivo real-time imaging [9–11]. Consequently, the physicochemical
properties of NPs open the possibility to obtain sensitive data in a nonin-
vasive manner, providing a breakthrough in early-stage cancer diagnosis,
⁎ Corresponding author.
E-mail address: miries@fata.unam.mx (M. Estevez).
stem cell tracking, drug delivery, and pathogen detection [12–18]. Several
types of NPs are currently being used for these applications, including me-
tallic NPs (Au and Ag) [19–21], quantum dots [22], magnetic NPs [23,24],
silica NPs [25,26], among other systems. In this line, silica NPs have gained
attention due to their properties in bioimaging, labeling and sensing [1,27,
28], as well as many reported advantages (including easy manufacture
and functionalization, high stability in different environments, biocom-
patibility and low cytotoxicity) [29–32]. Several strategies (e.g. covalent
binding, entrapment, and electrostatic interaction) have been developed
to formulate dye-doped silica NPs, for bioimaging, diagnosis, and thera-
peutic applications. For example, inorganic dyes [33], and/or enzymes
[34] have been used to functionalize silica NPs for some specific applica-
tions. In this regard, we report the functionalization of silica NPs using
fluorescent biocompounds extracted from “Palo azul” (Eysenhardtia
polystachya) (EP), in order to be used for biological applications as cell
entry biomarkers.
EP is a tree distributed in Mexico and Texas, and it is locally known as
“Palo dulce” (sweet wood), or “Palo azul” (blue wood). EP has been used
for the treatment of urinary tract and bladder infections, and as a diuretic
[35]. This tree gained attention due to its blue fluorescence as a result of its
infusion in water, and by its biological properties, including antidiabetic,
antibacterial, and antioxidant, among others [36,37]. Many studies have

http://dx.doi.org/10.1016/j.msec.2015.07.012
0928-4931/© 2015 Elsevier B.V. All rights reserved.
50 G. Ferreira et al. / Materials Science and Engineering C 57 (2015) 49–57
reported the principal components of EP extracted using methanol,
ethanol and water as solvents [35,36,38,39]. In this way, the responsible
compounds for the fluorescence of EP are Coatline A and Coatline B,
which represent a C-β-glucopyranosil-α-hydroxidihydrochalcone [40]
and different isoflavonoids [35]. It has been demonstrated that these
compounds are ionizable at physiological and basic pH values and fluo-
rescence is observed [35,36,39,41].
The aim of this work is to demonstrate how the fluorescent com-
pounds present in the EP Extracts (EPE), could be used in biomedicine
and biology applications. The reason to use them in those fields is be-
cause they fluoresce at physiological pH (6.8 - 8), then these compounds
could function as biomarker agents within biological systems.
Additionally, studies have reported that isoflavones have high
specificity to cells responsive to estrogen [42], increasing the potential
therapeutic effects of our novel system [43], which indeed not only
will work with high specificity to those cells acting as biomarkers, but
also, exert their estrogen-dependent biological activity.
In this work the formation of nanosystems from silica nanoparti-
cles and their functionalization using a luminescent extract from
“E. polystachya” has been reported. For the first the time, findings
that, these nanosystems may have crossed the MFC-7 cell membrane
have also been reported; we believe that the information and knowl-
edge obtained from this work would be important for the development
of novel nanosystems that could work as biomarkers/therapeutic
agents, and their suitable application within medicine, food and phar-
maceutical fields.
2. Experimental section
2.1. Materials
Methanol (MeOH), ethanol (EtOH) (90%), tetraethylorthosilicate
(TEOS) (98%), aminopropyltriethoxysilane (APTES), and potassium
bromide (KBr) were purchased from Sigma Aldrich. Chloride acid (HCl),
anhydrous ether (C4H10O), ammonium hydroxide (NH4OH) (28–30%),
phosphate buffer pH 4, and sodium hydroxide (NaOH) were purchased
from J.T. Baker.
Samples of EP were obtained from a local market, located in
Querétaro, México and were macroscopically and microscopically
indentified as E. polystachya (Ortego) Sarg. by Prof. Josefina Barajas, at
the “Xiloteca” (National Herbarium), Biology Institute, Universidad
Nacional Autónoma de México (UNAM). Samples were kept at the
National Herbarium under registration code MEXU 1850.
2.2. Extraction of compounds
Samples of EP heartwood were powdered using a grinder. EP powder
was subjected to soxhlet extraction using different solvents (methanol,
ethanol, methanol–ethanol, and methanol–ethanol–distilled water),
each independently. With a flow rate of 45 min/cycle and after 96 h of
extraction, the samples obtained were transferred into a separator funnel
in order to obtain different phases with anhydrous ether, and 5 M NaOH
solution. A precipitation reaction was reached changing the pH of the
solvent using low quantities of concentrated HCl. Samples were solubi-
lized with distilled water and dried at 60 °C for 4 h [44] to obtain four
powdered EP extracts (EPE), one for each solvent used.
2.3. Fluorescence of extracted compounds
The fluorescence of each EPE obtained was analyzed diluting EPE
powder in a phosphate buffer at different pH values (4, 7.4, and 8). An
UV–vis lamp was used to generate the fluorescence at two different
wavelengths, 254 nm and 365 nm. EPE with higher visual fluorescence
(EPEF) was chosen for further analyses.
2.4. Analytical detection of EPEF compounds
EPEF was characterized using a DT-Mini-2 UV–vis spectrophotometer
(Ocean Optics, Inc., Dudenin, FL, USA) in the wavelength range from 190
to 1100 nm, with a resolution of 1 nm. Briefly, EPEF was dissolved again in
methanol; then, methanol was continuously and controllably added to
adjust the concentration of the sample until the optical density of the
main absorption peak gave an absorbance reading in the region between
0.6 and 0.8 a.u. [45,46]. The acquired UV–vis spectrum was further ana-
lyzed using a fitted curve and data processing software FITYK 1.2.9 [47].
Furthermore EPEF was diluted in ethanol and the Shinoda reaction
was performed in order to confirm the presence of isoflavones. Briefly,
0.1 g of powder was diluted into 10 mL of EtOH. Magnesium pieces and
5 drops of concentrated HCl were added. After 5 min, a change of color
was observed. In order to assure the Shinoda results, EPEF (0.1 g) was
diluted in distilled water (10 mL), 5 drops of 5 M NaOH solution were
added, and the change of color was analyzed to determine the possible
compounds obtained in the extracts.
2.5. Development and functionalization of silica nanoparticles
2.5.1. Synthesis
Silica NPs were prepared by a modification to previously described
protocol [48]. Briefly, adequate quantities of MeOH/(EtOH), distilled
water, and NH4OH (20, 1, and 0.32 mL respectively) were mixed by
magnetic stirring during 5 min. Then, 5 mL of TEOS was added, and
the solution was magnetically stirred for 4 h at room temperature.
After time was completed, samples were dried at 120 °C in an Imperial
V laboratory oven (Lab-Line Instruments Inc., USA). Dried samples were
powdered in an agate mortar, washed 3 times using distilled water and
solvents (EtOH, and MeOH), and then stored in a free humidity container
until further use.
2.5.2. Functionalization
In order to enhance the interaction between EPEF and silica NPs, 1 g
of NPs was dispersed in ethanol and 5 mL of APTES was added; the
solution was refluxed at 120 °C under stirring for 24 h. Functionalized
NPs (NPs-APTES) were dried at 150 °C for 24 h, powdered and washed
with EtOH and distiller water.
One gram of NPs-APTES was dispersed in water acidified at pH 5, and
1 g of EPEF was added. The dispersion was magnetically stirred for 24 h
at room temperature. After this time, samples were dried at 80 °C for
24 h. Functionalized NPs (NPs-APTES-EPEF) were powdered and stored
in a free humidity amber container, for further use.
2.6. Physicochemical characterization
2.6.1. Particle size
Particle size distribution was determined by dynamic light scattering
(DLS), using a Brookhaven Instrument BI-220SM Research Goniometer
System equipped with a high-speed digital correlator PCI-BI9000AT, a
solid state photon detector and a He–Ne laser of 35 mW (633 nm),
using a Melles Griot 9167EB-1 as a light source (Brookhaven Instruments
Corp., USA). NPs were dispersed in EtOH, and measurements were made
at an angle of 90° for 10 s at 25 °C. For DLS analyses, NP samples were pre-
pared carefully by filtering from a colloidal dispersion, (previously placed
on a water-shaking bath at 20 °C and 60 shakes per minute to avoid par-
ticle aggregates), in a glass vial.
2.6.2. Electrical charge (zeta potential)
The zeta potential (ζ) of the synthesized NPs was measured by par-
ticle electrophoresis with an Acoustosizer IIS (Colloidal Dynamics, USA).
Samples were diluted in deionized water (≤18 Ω) in a concentration of
0.5% (w/v) and were placed into a plastic vessel.
 G. Ferreira et al. / Materials Science and Engineering C 57 (2015) 49–57
2.7. Morphology
The morphology of the NPs was analyzed by transmission electron
microscopy (TEM) (JEOL JEM-1010, USA). For TEM analyses NP samples
were prepared carefully by placing a drop of silica colloidal dispersion,
(previously placed on a water-shaking bath at 20 °C and 60 shakes per
minute to avoid particle aggregates), in a 300 mesh copper Formvar-
coated grid. Images were taken by using an acceleration voltage of 80
and 100 kV at different amplifications. Image analysis was performed
using ImageJ software 1.48 (US National Institutes of Health, Bethesda,
Maryland, USA).
2.8. The photoluminescence (PL) spectra
Raman spectra showing the PL signal were obtained at room temper-
ature using a micro-Raman spectrometer LabRAM II (DILOR) equipped
with a confocal optical microscope. Briefly, EPEF and NPs-APTES-EPEF
were diluted in a pH 7.4 phosphate buffer, then placed in a quartz cuvette
and PL was evaluated every 40 s during the first 6 min; then PL was eval-
uated every 5 min for 15 more minutes. Raman spectra were obtained by
excitation radiation at 480 nm (30 mW), an Ar-ion laser was employed as
excitation source; data were collected in the wavenumber ranges from
400 to 1000 cm−1. Silicon peak at 520 cm−1 was used as standard.
2.9. Infrared spectroscopy (FTIR)
Infrared absorption spectra of EPEF, silica NPs, NPS-APTES and
NPs-APTES-EPEF were examined by FTIR spectroscopy (Bruker, Vector
33, Biospin Corporation, USA). Dried samples with a minimal moisture
content, were mixed with KBr. Spectra were collected with a wavenum-
ber range of 400 to 4000 cm−1.
2.10. Biological application of functionalized nanoparticles
2.10.1. Cell internalization assay
In order to analyze the ability of the fluorescent functionalized nano-
particles to internalize into MCF-7 cells (ATCC), studies were carried out
following a previously reported protocol [49]. Briefly, MCF-7 cells were
grown according to the manufacturer's instructions in an air-CO2 atmo-
sphere at 37 °C. Cells were seeded into 3 cm Petri dishes. Cells were
treated with 4% NPs-APTES-EPEF suspension for 48 h. After incubation
time, cells were washed with PBS and the internalized NPs were
observed under the confocal microscope at 20× (LSM510 Zeiss).
3. Results and discussions
3.1. Fluorescence of extracted compounds
Fluorescence results showed that EPE obtained using EtOH, at
254 nm and 365 nm wavelengths, presented higher fluorescence than
compounds extracted with any other solvent (data not shown); hereinaf-
ter this extract will be referred as EPEF. In Fig. 1, EPEF fluorescence at
different wavelengths (254 nm and 365 nm) is shown; it seems that at
neutral–basic pH, molecules extracted are ionizable and the fluorescence
Fig. 1. Fluorescence of EP extracted compounds in ethanol determined at (A) 254 nm and (B) 365 nm.
51
is observed, as it has been mentioned by other authors [41]. Therefore,
extracted compounds using EtOH (Ext-EtOH) were used for further anal-
yses presented in this work.
Fluorescence can be attributed to the presence of Coatline B into the
extract [40]; according to Acuña [50] higher ionization of the Coatline by
the medium initiates the transformation from Coatline B to Matlaline
being this compound responsible for fluorescence.
3.2. Analytical detection of EPEF compounds
As mentioned before in the Experimental section, EPE was subjected
to precipitation reaction by changing pH; with this, it was assured that
only polyphenolic compounds were precipitating and the presence of
flavonols, isoflavonoids, and chalcones at EPEF was successfully detected
with the preliminary test of Shinoda and NaOH (Table 1) according to
[51–53].
UV–vis scan was performed in order to determine the presence of
flavonoids at EPEF. Fig. 2 shows a comparison between the original
spectrum from EPEF with a curve modeled spectrum obtained with
two overlapping peaks (convolved); the best fit was obtained with
two Gaussian centered on 283 nm and 385 nm, with heights of
0.429 and 0.272 respectively; this allowed us to state that the UV–vis
spectrum showed the two characteristic bands for flavonoid skeleton,
at 240–280 nm for band II and at 320–380 nm for band I [54–56]. These
results confirmed that EPEF (ethanolic extract) contains isoflavones,
flavones and flavonols.
3.3. Size, electrical properties (ζ), and morphology
We evaluated the physicochemical properties of all silica-based
nanoparticles obtained in this work in order to elucidate the functional
performance. Properties such as size, electrical charge, morphology and
physical state were studied. We therefore measured the mean particle
diameter and ζ-potential of non-functionalized and functionalized
silica-based nanoparticles (Table 2). We obtained very small silica NPs
by DLS, with average diameter ranges from 43 to 175 nm.
The functionalization of silica nanoparticles with APTES and
afterwards with EPEF affected the NPs size, turning size from 43 nm,
to 113 nm and 175 nm (NPs, NPs-APTES, and NPs-APTES-EPEF, respec-
tively). NPs-APTES standard deviation of 32.5 nm was recorded due to
the agglomerates that formed quickly into the colloidal system, this
was also observed in the histogram corresponding (Fig. 3B), where
the size of about 45 nm was recorded; above 45 nm modes statistics
were the result of agglomerations between nanoparticles which domi-
nated the computer accounts. It seems that the presence of organic sur-
face groups, especially amino containing residues, as APTES, and the
combination of APTES and EPEF influenced the increase of particle
size, by increasing the surface of the silica NPs previously developed.
It seems that organic compounds attached to the surface of the nano-
particles increase the electrostatic repulsion of the silica tails at the sur-
face, increasing in a few nanometers the particle size. Our findings
confirm the results by other authors [57–60]. We demonstrated by
standard deviation values that synthesized NPs and NPs-APTES-EPEF
had a homogeneous diameter, see Table 2 and Fig. 3. For functionalized
52 G. Ferreira et al. / Materials Science and Engineering C 57 (2015) 49–57
Table 1
Results obtained by Shinoda test on each extract from Eysenhardtia polystachya (Ortega) Sarg.
Solvents Appearance before Shinoda test
Ethanol Slightly yellow
Methanol Colorless
Metahol/ethanol Colorless
Metahol/ethanol/water Colorless
particles of APTES-EPEF; the standard deviation and high wide range
size can be reasonably explained by fast agglomerate formation which
in turn can be explained by the ζ-potential of these particles, also
discussed below.
ζ-Potential analyses were evaluated at pH 7.4, which corresponds to
the physiological pH. The main idea of these analyses was to determine
the possible biomedical applications of these functionalized NPs in a
near future. Results showed that non-functionalized silica NPs had an
electrical charge of −20.1 mV, which could be attributed to the electro-
negativity of the silanol groups (Si–OH) at the surface of the NPs after
the synthesis process. When silica NPs were functionalized using
APTES and APTES-EPEF, ζ-potential shifted from negative values to
positive values (+ 12.9 mV and + 8.5 mV, respectively), indicating
that NH+ 2 groups were located on the silica NP surface. Then, in NPs-
APTES-EPEF, there was a decrease in the ζ-potential, from + 12.9 mV
to + 8.5 mV, which was related to the presence of EFEF coupled to
APTES on the surface of the NPs. These results confirmed that modi-
fication of silica NP surface was reached in a very easy way. However,
ζ-potential values obtained indicate that non-functionalized and
functionalized silica NPs were close to their isoelectric point (IP), which
would lead to aggregation in a colloidal solution or other physiological
fluids, mainly by their weak electrostatic repulsion. Nevertheless, the
low electrical charge in the surface of the functionalized nanoparticles
allowed the internalization of these carriers into human cells. Some
studies have encountered that zeta potential of some normal and cancer
human cells has negative values (between −20 and −30 mV) [61]. Thus,
the opposite charges on the surface of functionalized nanoparticles
(+8.5 mV) and the negative charge of human cells could increase the in-
teractions between them, enhancing the entry process into the cell ruled
by electrostatic attractions. Therefore, the ability of our functionalized
NPs to act as biomarkers and/or drug delivery systems was confirmed
using parameters like ζ-potential, due to an increase in their NP–cell in-
teraction properties.
Morphology of NPs, NPs-APTES, and NPs-APTES-EPEF was deter-
mined by TEM (Fig. 4). Micrographs showed that particles have irregular
spherical shapes with a mean size of 20.08 ± 2.79 nm; 97.4 ± 14.81 nm;
and 118.71 ± 7.36 nm for NPs, NPs-APTES, and NPs-APTES-EPEF
Fig. 2. UV–vis scan of flavonoids extracted from EP.
Appearance after Shinoda test Possible chemical compound present
Yellow Flavones and flavonols or isoflavones
Colorless Isoflavanones chalcones and aurones
Colorless Isoflavanones chalcones and aurones
Colorless Isoflavanones chalcones and aurones
respectively. Diameter size differences between DLS and TEM are reason-
able for the fact that the hydrodynamic diameter is usually higher than
the diameter obtained by direct analysis to the particle using TEM images.
Additionally, as could be seen (Fig. 4), agglomerates of nanoparticles
were formed of NPs and NPs-APTES contrary to what was observed with
NPs-APTES-EPEF; there, the nanoparticles were observed perfectly
formed. These results are consistent with the DLS results discussed on
above lines; for NPs, it can be explained by hydrogen bridge between
surface groups (–OH); for NPs-APTES, it can be explained by weak
attractive intermolecular forces between the negative surface areas
and positive surface areas due to (–OH) survivor groups and (–NH2)
emergent groups.
TEM results confirm that non-functionalized and functionalized silica
particles have the appropriate size to be considered NPs; also these
results prove that functionalization of silica NPs affects the size (due to
the presence of the components used for functionalization) but not the
shape.
3.4. FTIR characterization
As we showed in the ζ-potential data, the electrical charge of func-
tionalized silica NPs was affected by the presence of APTES or APTES/
EPEF. These shifted charges indicate that some interactions were formed
in order to create the conjugation among silica NPs-APTES-EPEF. FTIR was
used to elucidate the molecular interactions among components of our
novel system (Fig. 5).
FTIR spectra of silica NPs showed a number of characteristic absorp-
tion bands: O–H stretching in H-bonded water (at 3700–3000 cm−1;
centered on 3462 cm−1), asymmetric vibration of Si–O (1100 cm−1),
asymmetric vibration of Si–OH (960 cm−1), and symmetric vibration of
Si–O (795 cm−1). Also this band can be cross checked through the
1635 cm−1 band due to scissor bending vibration of molecular water.
The absorption bands between 800 and 1260 cm−1 have been described
as a superimposition of various SiO2 peaks, Si–OH bonding and peaks due
to residual organic groups. Besides, the absorption bands 2980 cm−1
(CH3) and 2930 cm−1 (CH2) can be used to identify the presence of
unreacted TEOS in the silica NPs. The presence of these bands is in agree-
ment with findings of previous studies [62]. For APTES functionalized
silica NPs, the adsorption bands in the range of 3700 to 3000 cm−1 are
due to the stretching vibrational frequency of the silanol groups. How-
ever, during the surface functionalization, the surface hydroxyl groups
react with the APTES to form Si–O–Si bonds. This reduction in the hy-
droxyl groups suggests an anchoring mechanism involving a reaction
between the silanol groups and APTES. Thus, the peak at 941 cm−1 is
decreased, due to the conjugation of silica NPs with APTES, where the
silanol groups have been diminished. Therefore a new band assigned
to NH2 asymmetric bending was observed at 1590–1560 cm− 1. The
band of C–H bonding (at 2930 cm−1) almost disappeared, suggesting
that there was a possibly deacetylation degree of C–H bonds. These re-
sults confirm a successful functionalization of the silica NPs, and also,
our results are in agreement with the results previously reported by
other authors [62–67]. The FTIR spectra of NPs-APTES-EPEF showed
some changes in the characteristic absorption bands showed for non-
functionalized silica NPs, and NPs-APTES. First, between 2800 and
3700 cm−1, the absorption band for amine groups (NH stretch) was
not found, Instead, a broadened band, possibly attributable to phenolic
hydroxyl groups from EPEF which dominates this region, suggests that
 G. Ferreira et al. / Materials Science and Engineering C 57 (2015) 49–57 53

Table 2
Mean particle size, standard deviation, and electrical charge (ζ) of non-functionalized and functionalized silica-based nanoparticles.

Nanoparticles Number data total Average size (nm) Minimum size (nm) Median size (nm) Maximum size (nm) ζ-Potential
(mV at pH 7.4)

Si (non-functionalized) 243 43.13 ± 3.56 35 43 48 −20.1 ± 7.8a

APTES functionalized 243 113.08 ± 32.47 45.72 128.32 148.31 +12.9 ± 1.3b
APTES/EP-F 243 175.21 ± 2.06 171 175 180 +8.5 ± 1.4b

Results are expressed as mean ± S. D. of three repetitions. In zeta potetial column, different letters indicate significant difference (P b 0.05) between rows.

OH-bonding was present between EPEF compounds and APTES func-
tionalized silica nanoparticles. On the other hand, between 800 and
1800 cm−1, the absorption band of –NH2 (at 1590–1560 cm−1) previ-
ously found in the NPs-APTES spectrum, here, was not found, which in-
dicates the successful conjunction between NH2 groups from APTES and
EPEF compounds. Similar findings, were observed previously by anoth-
er authors [68,69], where they found that flavonoids, such as quercetin
and catechin, were successfully incorporated and retained into poly-
meric NPs, mainly by OH bonding and interactions between carbonyl
groups from polymer and flavonoids. Thus, these results indicate that
EPEF compounds were coupled to APTES functionalized in an effective
way.
3.5. Photoluminescence of EPEF compounds and NPs-APTES-EPEF
It has been demonstrated that the presence of atmospheric oxygen
causes photodegradation in many flavonoids, decreasing their biological
properties, including their luminescence/fluorescence [70–72]. In this
regard, some authors have claimed that the dye performance can be
affected when it is attached to the surface of carriers, e.g., it causes spec-
tral shifts of both absorption and emission, it affects photostability, and it
alters the fluorescence lifetime [73–76]. Therefore, Raman spectroscopy
was used to evaluate photo-stability of EPEF compounds (free or attached
to the NPs-APTES surface). Fig. 6a summarizes the PL EPEF properties;
emission peak was centered in 545 nm, showing that after incubation
the PL of EPEF decreased through time. This indicates that the EPEF
compounds were oxidized by molecular oxygen from the environment.
However, when EPEF was complexed with silica NPs, the photostability
was maintained (Fig. 6b), this behavior is not yet well understood but
here it is clear that the interaction between EPEF with NPs-APTES im-
proves the PL stability; it is possible that this interaction caused that an
amount of π-electrons were leased to higher intramolecular electronic
transfer [77]. When comparing EPEF with NPs functionalized and
APTES/EPEF, a Δλ ≈ 7 nm blue shift was observed for functionalized
NPs (Fig. 6b) which we attribute to the NPs. Other authors have reported
similar shifts when fluorescent molecules were confined within porous
particles [78–81]; they also attribute the blue shift to confining effects,
in our case the EPEF compounds were not confined within the pores,
on the contrary, they were anchored on the surface as shown in the
“size, electrical properties (ζ), and morphology” data, therefore, there
was no doubt that confining effect of molecules was not the only effect in-
volved in the blue-shifting in PL spectra.
3.6. Cell internalization assay
NPs-APTES-EPEF internalization was studied using MCF cells and
confocal laser-scanning microscopy (CLSM) as shown in Fig. 7; the mi-
crographs of CLSM on MCF-7 breast cancer cells using EPEF compounds
alone are shown in Fig. 7a and b in a dark field and bright field respec-
tively, and on the other hand the NPs-APTES-EPEF system is shown in
Fig. 7c and d, dark field and bright field respectively. In all cases, MCF-7
cells were incubated with EPEF compounds or with the NPs-APTES-
EPEF system for 48 h cells were observed using a Zeiss laser scanning con-
focal microscope.
In Fig. 7b (bright field micrograph) MCF-7 cells were seen, however
in Fig. 7a (dark field micrograph) MCF-7 cells were not seen, because
the EPEF compound luminescence during the 48 hour incubation have
been decayed. In contrast, in Fig. 7c (dark field micrograph) the MCF-7

Fig. 3. DLS measurements for (A) silica NPs; (B) NPs-APTES; (C) NPs-APTES-EPEF; and (D) comparison of previous distributions.
54 G. Ferreira et al. / Materials Science and Engineering C 57 (2015) 49–57

The internalization of the functionalized NPs (NPs-APTES-EPEF)

within tumor cells is the hallmark of this study to evaluate the potential
biomedical application of this biomarker.

4. Conclusions

Ethanol extract (EPEF) from Palo azul, identified at the National

Herbarium, consists of flavones, flavonols and isoflavones. These extract
exhibit fluorescence at physiological pH and using a modified Stöber
method, we were able to obtain spherical silica nanoparticles which
were functionalized. At pH 7.4, silica NPs functionalized with EP extract
are close to their isoelectric point, suggesting a net charge which allows
the functionalized NPs to cross the cell membrane.
According to the results obtained by DLS and TEM images, it can be
concluded that a high density of anchors was on the surface of NPs,
otherwise it would not be easy to record an increase in size as the one
achieved. This is an important indicator because it predicts a high bio-
availability of the biomarker, also low or no toxicity is foreseen because
both compounds (NPs and ethanolic extract) are GRAS substance, these
characteristics are important even though we performed an in vitro assay.
From the results of DLS, TEM and IR we deduced that the anchor be-
tween the NPs and the compounds of ethanolic extract is real, but it is dif-
ficult to conclude about the chemical or physical interactions that
predominate in this anchorage. So a thorough study focused on resonance
spectroscopic techniques such NMR is justifiable and recommended.
The data obtained from PL and CLSM demonstrate the feasibility of
the extract from E. polystachya using ethanol as extraction medium to
be used as a biomarker in breast cancer cells, once functionalized with
silica NPs, an enhanced bioavailability was assured, resulting in a poten-
tially therapeutic agent.

Abbreviations
NPs nanoparticles
EP Eysenhardtia polystachya
EPE Eysenhardtia polystachya extracts
EPEF Eysenhardtia polystachya extracts with fluorescence
APTES aminopropyltriethoxysilane
NPs-APTES silica nanoparticles functionalized with
aminopropyltriethoxysilane
NPs-APTES-EPEF nanoparticles functionalized with fluorescence
extract from Eysenhardtia polystachya
MCF-7 breast cancer cell
CLSM confocal laser-scanning microscopy
MeOH methanol
EtOH ethanol
Fig. 4. Micrographies of (A) silica NPs; (B) NPs-APTES; and (C) NPs-APTES-EPEF (obtained TEOS tetraethylorthosilicate
by TEM), showing spheric shape and a small particle size. DLS dynamic light scattering
TEM transmission electron microscopy
PL photoluminescence
FTIR Fourier transform infrared spectroscopy
ATCC ATCC organization
cells were visualize by the luminescent of the NPs-APTES-EPEF system PBS phosphate buffered saline
working as a cell dye; green staining demonstrated the presence of
the dye (NPs-APTES-EPEF nanosystem) with the cells found in bright Acknowledgment
field micrograph (Fig. 7d), and dark field micrograph (Fig. 7c) demon-
strated the shape and limits of each cell found in bright field micro- The authors are grateful to MC. Josefina Barajas for her expertise in
graph. Therefore MFC-7 cells treated with EPEF alone did not show the E. polystachya sample identification; to MC. Lourdes Palma-Tirado
cellular uptake, however cells treated with NPs-APTES-EPEF showed in- for her TEM support; to Eng. Francisco Rodríguez Melgarejo for his
ternalization by the tumor cells, indicating that NPs-APTES-EPEF was expertise in the microscopy Raman and his knowledge and support;
suitable to enter the cell without any further means of permeabilizing to the undergraduate students Andrea Prado-Trillo and Alonso Castillo
the cell membrane, and will be able to elicit a biological activity, which re- for their dedication to prepare samples and support as necessary to
mains to be elucidated This finding represents a breakthrough approach carry out the project; to Eng. Bernardino Rodríguez-Morales and MC.
to biomedical research; in single functionalized NPs we have a fluores- Guillermo Vázquez-Sánchez for their technical support to carry out
cent biomarker and a possible therapeutic agent for breast cancer cells the project and revision the project; and Eng. Ana L. Ramos-Jacques
estrogen-dependent cells. for her comments as proofreader.
 G. Ferreira et al. / Materials Science and Engineering C 57 (2015) 49–57 55

Fig. 5. FTIR spectra of non-functionalized silica nanoparticles (NPs), APTES-functionalized silica nanoparticles (NPs-APTES), and APTES/EPEF functionalized silica nanoparticles
(NPs-APTES-EPEF).

Fig. 6. Raman spectrum of the photoluminescence of free fluorescent EP-F (a) extracted with ethanol at pH 7.4 and (b) EP-F attached to silica nanoparticles by Raman spectroscopy.
56 G. Ferreira et al. / Materials Science and Engineering C 57 (2015) 49–57
Fig. 7. CLSM micrographs for EP isoflavones alone (a and b), where no cellular uptake is shown and for functionalized nanoparticles (c and d) where cellular uptake is confirmed.
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