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Structural, luminescence and geno/cytoxicity

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study of carbon dots derived from

Cite this: DOI: 10.1039/c9nj03771c
Opuntia ficus-indica (L.) Mill
Eduardo Madrigal-Bujaidar, a Genaro Ivan Cerón-Montes, b
Joan Reyes-Miranda, c Erasto Vergara-Hernández, d Isela Álvarez-González, a

Ángel de Jesús Morales-Ramı́rez, e Luis Enrique Francisco-Martı́nez b and

Aristeo Garrido-Hernández *b

Received 20th July 2019,
Accepted 8th November 2019
DOI: 10.1039/c9nj03771c
rsc.li/njc
Photoluminescent carbon dots (CDs) were synthesized by the hydrothermal method. In the synthesis,
different plants as carbon sources were used. Photoluminescence results demonstrated the highest
emission for CDs coming from nopal. Therefore, different concentrations of nopal during the CD syntheses
were evaluated to determine the maximum emission intensity. Transmission electron microscopy revealed a
narrow particle distribution (5–7 nm). Furthermore, the indexed planes (213) and (203) demonstrated the
hexagonal phase of these CDs. Chromatic coordinates x = 0.157 and y = 0.158 confirmed blue emission
according to the CIE diagram. The genotoxic and cytotoxic potential of nopal CDs were evaluated with the
micronucleus test in mice, and with the relationship between young and mature erythrocytes, also in mice.
Five doses of CDs were tested by the intravenous route (0.02, 0.2, 2, 25, and 50 mg kg 1). Results obtained
in micronuclei showed that all tested doses were genotoxic, except the two low doses at 96 h of the assay.
With respect to cytotoxicity, the CDs also induced a bone marrow proliferation decrease along the assay,
particularly 48 h after exposure. Thus, our geno/cytotoxicity assay revealed that the CDs were able to
damage DNA and to cause cytotoxicity, and also suggested that biomedical approaches can be preferable
assayed at the lowest tested dose range (0.02 mg kg 1).

1. Introduction in 2004.6 CDs have shown excellent biocompatibility, which allows

Carbon dots (CDs) or carbon nanoparticles with a size less than
10 nm have been attracting attention since their discovery
because of their interesting physical and chemical properties
such as facile functionality, absence of blinking, tunable photo-
luminescence, and low photo-bleaching,1–3 characteristics that
can even be superior to traditional semiconductor quantum dots
and organic dyes.4,5 Fluorescent carbon dots were obtained for the
first time during purification of single-walled carbon nanotubes
a
Laboratorio de Genética, Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional, Wilfrido Massieu s/n, Zacatenco, CP 07738 CDMX, Mexico
b
Universidad Tecnológica de Tecámac, UTTEC, Carretera Federal México, Pachuca
Km 37.5, CP 55740, Col. Sierra Hermosa, Tecámac, Estado de México, Mexico.
E-mail: agarridoh@uttecamac.edu.mx
c
Universidad Autónoma Metropolitana Azcapotzalco, Departamento de Materiales,
Av San pablo 180, Col Reynosa-Tamaulipas Azcapotzalco, CP 02200, CDMX,
Mexico
d
Instituto Politécnico Nacional, UPIIH, San Agustı́n Tlaxiaca, CP 42080, Hidalgo,
Mexico
e
Instituto Politécnico nacional-ESIQIE, Av. Instituto Politécnico nacional s/n,
CP 07738, CDMX, Mexico
them to possess great potential in biological applications, with a
performance comparable to or even better than conventional
quantum dots.7–11 Properties of CDs previously described provide
these nanoparticles with a wide range of applications including
the construction of sensors,12–14 photoelectric devices,15 and
applications in photocatalysis,16–20 phototherapy,21 and bio-
imaging.22,23
Photoluminescence is one of the most interesting properties
of CDs and although the mechanism involved in this property
is not completely clear,24 successful applications of this nano-
material, such as a fluorescent probe to visualize biological
systems both in vitro and in vivo, have been developed.25–27
For obtaining fluorescent carbon quantum dots, several top
down approaches have been proposed. However, they require
toxic reagents and special equipment since they involve a non-
selective exfoliation process. Syntheses of CDs have been
reported by using acid oxidation, electrochemical synthesis,
arc discharge, laser ablation, microwaves and ultrasonic waves,28
most of which require complex conditions during their synthesis,
long post-treatment times or expensive carbon sources, which
limit their applications. On the other hand, among the bottom up

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 New J. Chem.

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approaches, a hydrothermal process has been used as a suitable
system for preparing highly luminescent CDs, involving carbo-
nization of glucose, sucrose, glycol, glycerol, or citric acids,
among other compounds.27 In this context, interestingly,
S. Swagatika et al.29 reported a simple one-step synthesis to
produce highly luminescent CDs from orange juice by using the
hydrothermal method.
Although it is a challenging task, there has been much effort
in the research community directed towards utilizing renewable bio-
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precursors to prepare CDs using inexpensive and biocompatible
precursors, such as garlic,24 honey,30 papaya,31 limeade,32 bamboo
leaves,28 pomelo peel,33 lemon34 and ginger,35 which are herbaceous
plants, fruit beverages, and plant sources which have greatly
aroused interest because of their green chemical nature.
Furthermore, the use of biological markers in the evaluation
of risk for diseases of mutational etiology has increased markedly
in the last decade.36 In this context, the studied nanoparticles are
particularly useful in the evaluation of progressive diseases
that manifest their symptoms long after exposure to initiating
factors.8–10 Therefore, at the present state of research on the
matter evaluating the capacity of CDs to affect genetic material
is relevant. In this field, various studies to determine geno/
cytotoxicity have been published with variable results;37 they
were performed mainly in aquatic organisms, cultivated cells,
and in in vivo systems to a lesser extent. The micronucleus test
is included in most genotoxic guidelines because it has been
validated as a useful tool to determine the genotoxic potential
of numerous physical and chemical agents, and moreover
because it is a versatile test that can be applied in in vitro
and in vivo assays, and because such a parameter has been
correlated with the presence of cancerous cells, and with the
presence of other chronic diseases.38,39 In this context, the
knowledge that electrochemically produced CDs can generate
reactive oxygen species including O2 (singlet oxygen) and OH
after exposure to blue light is interesting because this type of
activity may not only explain the observed genotoxicity, but may
also suggest CDs’ applicability to kill cancer cells.1,4,5 According
to the indicated information, the present report was designed
with two main objectives. The initial objective was to carry out
the synthesis and characterization of CDs from various plants
prepared through the hydrothermal approach; among these
plants, Opuntia ficus-indica (L.) Mill, a native Mexican plant
better known as nopal, was used. According to the best of
our knowledge there are no reports about carbon dots derived
from nopal. In this vein, nanoparticles were synthetized by
the indicated method, and characterized by means of Fourier
Transform Infrared Spectroscopy (FT-IR), Transmission Elec-
tron Microscopy (TEM) and photoluminescence spectroscopy.
Secondary metabolites belonging to nopal which might cause
genotoxicity and cytotoxicity were also tested in the CDs by
phytochemical screening. Our second aim was to determine
the genotoxic and cytotoxic potential of CDs obtained from
nopal, since this plant showed the highest photoluminescence
intensity. For such a purpose, we examined the induction of
micronuclei in mice, as well as their effect in bone marrow cell
proliferation.
New J. Chem. This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
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2. Experimental section
2.1 Synthesis of carbon dots
Guava (Inga feuilleei), nopal (Opuntia ficus-indica), cucumber
(Cucumis sativus), and avocado (Persea americana) were purchased
in the local mall and used without any previous purification. The
vegetables were initially washed and sun-dried in the same
environmental conditions. Subsequently, 0.75 g of the dried
vegetables were cut into small pieces and mixed with 30 mL of
distilled water; the mixture was then crushed homogeneously by
using a conventional blender, and poured into a Teflons lined
stainless-steel autoclave with a capacity of 45 mL. The autoclave
was placed inside an oven and heated at 160 1C for 12 h. Following
the hydrothermal process, the appearance of the mixture turned
from colorless into a dark yellow hue; such a process involved the
reduction of plant sources into carbon nanomaterials. The dark
yellow solution was separated by centrifugation at 6000 rpm for
30 min. Then, the supernatant was vacuum filtered in a 2.7 mm
paper filter, and a yellow aqueous suspension was finally obtained.
2.2 Characterization of the CDs
The morphology and the crystalline structure of each CD were
elucidated by using high resolution transmission electron
microscopy (HR-TEM), JEM-200FS, operating between 80 and
200 keV. The emission and excitation spectra of the samples
were recorded on a Hitachi F-7000 fluorescence spectrophoto-
meter. Infrared spectra were obtained at room temperature with a
PerkinElmer Spectrum Two spectrophotometer. The spectra were
recorded with 32 scans in the 4000–450 cm 1 range.
The carbon dot concentration was obtained by determining
the weight of carbon dots in powders. For this purpose, 20 mL
of carbon dot solution was dried (at 50 1C, 0.7 Pa and 12 h) in
a vacuum-freeze dryer (Labconco, FreeZone 2.5 L).
2.3 Qualitative phytochemical screening
Phytochemical screening detection was performed as follows.
Tests for alkaloids using Wagner’s test. To 5 mL of 10 wt%
hydrochloric acid 0.5 mL of CDs were added, and the solution
was heated for 5 minutes. After that the solution was cooled
down and filtered, and finally a few drops of Wagner’s reagent
were added.
Detection of flavonoids. 0.5 mL of CDs were dissolved in 2 mL
of ethanol. Using the Shinoda reaction test, 2 drops of concentrated
hydrochloric acid were added to the solution. In addition the
sodium hydroxide test was performed; for this purpose 3 drops
of 10 wt% sodium hydroxide solution were added to the CDs.
Test for phenolic compounds using the ferric chloride test.
25 mL of CDs were diluted with 50 mL of distilled water. A few
(1 to 4) drops of neutral 5% ferric chloride solution were added
to the solution.
Test for saponins. 1 mL of CDs were shaken for 15 minutes;
the presence of 8 mm or 10 mm of foam indicated the presence
of saponins. In addition, the Lieberman Bouchard reaction was
performed. 0.5 mL of CDs were concentrated in 0.2 mL, there-
after, 2 drops of acetic anhydride were added and esterified using
2 drops of concentrated sulfuric acid.

NJC
Test for tannins. 2 mL of distilled water and 3 drops of
2 wt% sodium chloride were added to 1 mL of CDs. The
resulting solution was heated until boiling for 1 minute. 5%
ferric chloride solution was added to 2 mL of the test solution.
All tests were carried out in triplicate.
2.4 Genotoxic and cytotoxic potential of nopal carbon dots
For these two purposes we used male mice obtained from the
bioterium of the Hidalgo State Autonomous University. The
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animals had a mean weight of 25 g. They were maintained in
polypropylene cages at 23 1C, 40% relative humidity, and a 12 h
dark–light cycle. They were conditioned in the animal house of
the Genetics laboratory for a week before starting the assay.
The mouse in vivo protocol was carried out according to the
Mexican norms: NOM-033-ZOO-1995 for the animal sacrifice
and NOM-062-ZOO-1999 for the care and use of laboratory
animals. Such norms agree with the Legislation for Protection
of Animals used for Scientific Purposes (Directive 201/63/EU).
Moreover, the applied protocol was approved by the Institu-
tional Animal Care and Use Committee of the Autonomous
University of Hidalgo State (México).
For the geno/cytotoxic assays, six mice per group were intra-
venously (iv) injected once and organized as follows: a control
group treated with 0.4 mL of injectable water, a positive control
group that was administered with doxorubicin (1.5 mg kg 1),
and five groups that were administered CDs in doses of 0.02, 0.2,
2, 25 and 50 mg kg 1.
Before administering the mentioned substances, we took a
peripheral blood sample from the tail of each mouse and extended
it on two clean slides, and then the cells were fixed in absolute
methanol for 5 min, washed in running water, dried, and stained
with 5% Giemsa solution made in phosphate buffered saline
(pH of 6.8) for 12 min. All animals were then placed in their
corresponding cages for the next 96 h. During this period, and
through a procedure similar to the one described earlier, the
peripheral blood of each mouse was obtained, fixed, and stained
at 24, 48, 72, and 96 h post-treatment.
The experimental study and microscopic observations were
performed according to previous reports on the matter.40,41 Deter-
mination of the genotoxic potential of the CDs included the
quantification of micronucleated polychromatic erythrocytes (MNPE)
in 1000 polychromatic erythrocytes (PE) per mouse. Besides, to
examine the bone marrow cytotoxic potential of the nanoparticles,
we determined the proportion of PE with respect to the number of
normochromic erythrocytes (NE) in 1000 erythrocytes per mouse.
The statistical significance of the obtained results was determined by
means of an ANOVA followed by a post hoc Student–Newman–Keuls
test using the SigmaPlot software, version 12.1.
3. Results and discussion
3.1 Photoluminescent properties and Raman spectroscopy of
the CDs
The emission and excitation spectra of CDs prepared from
guava, nopal, cucumber and avocado are shown in Fig. 1a
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Fig. 1 (a) Excitation and (b) emission spectra of CDs obtained from
different carbon sources.
and b, respectively. The emission and excitation spectra exhibit
different luminescent intensity, suggesting that the composition
of the fruits and vegetables used as the carbon source might have
affected the luminescent properties. Fig. 1a reveals that the
strongest maximum intensity among the four tested samples
corresponds to the CDs obtained from nopal, when they were
excited at a 375 nm wavelength. In general, all the excitation
spectra presented their maximum excitations in a wavelength
range between 370 nm and 390 nm, where the largest number of
particles were excited, whereas their maximum emission was
registered in the wavelength range between 440 nm and 490 nm
(2.83–2.63 eV).
Our findings suggested that the particular composition of
each plant used as the carbon source might change as a result
of agronomic, genetic, environmental and physiological factors.
Related to this point, Table 1 shows the general composition of
the fruits and vegetables used in the study as the carbon source to
obtain the CDs, including the weight percentage of fat, proteins,
carbohydrates and water, factors that show the principal compo-
sition of the used fruits and vegetables.
According to the observed results, the CDs obtained from
avocado presented the lowest emission intensity. This finding
suggests that the fat amount is an important parameter to be
considered during the hydrothermal synthesis, since avocado
has the highest fat amount (around 23.5 wt%). Fat might
promote the formation of carboxylic groups; the presence of
this group produces several surface defects, which can act as
excitation traps, reducing the luminescence intensity due to the
increase of non-radiative recombination on the surface of the
carbon dots induced by carboxyl radicals (see Fig. 1b).42,43
Besides, Bui et al.44 demonstrated that carbon sources with
small amounts of fat and proteins offer high luminescence
Table 1 Components of the carbon sources used during the carbon dot
synthesis
Carbon source Fat Proteins Carbohydrates Water
Nopal 0.1 1.4 3.3 91
Cucumber 0.5 1.8 2 95
Guava 0.5 0.8 5.8 78
Avocado 23.5 1.8 0.4 75
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Paper NJC

intensity, where any carbohydrates containing C, H and O in a
ratio 1 : 2 : 1 are adequate to grow carbon quantum dots from
hydrothermal conditions.
On the other hand, the composition of the carbon source
played at important role in the carbonization process; as a
result different concentrations of carbon dots were obtaining
under the same synthesis conditions. The concentrations of the
carbon dots were 0.83, 0.81, 0.75 and 0.73 mg mL 1 for nopal,
cucumber, guava and avocado, respectively. Although the guava
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were raised. Also, different behavior was found by varying the
nopal concentration in the CD syntheses, since the PL intensity
of CDs obtained at 100 mg mL 1 was lower than that of CDs
prepared at 25 mg mL 1 keeping constant the reaction time
and temperature (2.5 h and 200 1C, respectively). These results
suggested that 25 mg mL 1 is a better concentration than
100 mg mL 1, since, according to the literature, the intensity
of the PL spectra sharply increases by lessening the concen-
tration of CDs due to the interaction among the different polar

carbon dots presented a similar concentration to that of the groups at low concentrations.
avocado counterparts, their luminescent intensity was higher. Otherwise, the PL intensity decreases as a result of CD
This can be explained by the carbon dot surface, which is highly agglomeration caused by the high concentration of nanoparticles.
influenced by the composition of the carbon sources. Nevertheless, an optimum concentration has not really been
The mechanism of the photoluminescence (PL) behavior of reported since it depends on various factors, such as the carbon
CDs is very complicated and has not yet been clearly under- sources used to obtain the CDs, and the mechanism to form
stood. The PL intensity depends on the number of particles them. Based on our results, 25 mg mL 1, 12 h and 200 1C were
excited at a particular wavelength. The PL behavior is due to the optimum synthesis conditions for preparing CDs. Bibekananda
carbon dot particle sizes and their surface defects, which are and Niranjan48 found a decrease in the luminescence intensity for
inherent to their synthesis conditions and the plant used as banana carbon dots for concentrations higher than 0.02 mg mL 1.
the carbon source.45–47 Since the CDs obtained from nopal In this work, the ‘‘optimum concentration’’ was 1.2 mg mL 1;
presented the highest emission intensity, different synthesis normally the best carbon dots concentration for reaching a high
conditions were applied in order to obtain the optimum emission luminescent intensity differs from each other due to the carbon dot
intensity (Table 2). size, synthesis conditions, and carbon sources. Higher temperature
In our study with nopal, it seemed clear that PL intensity of promotes the degree of the carbonization reaction, which induces
the obtained CDs depended on the synthesis conditions as better luminescent intensity as demonstrated by Lin et al.;49 they
shown in Fig. 2. In the figure, our results showed that the PL also showed that an excessively high temperature of the reaction
intensity increased when the temperature and the reaction time will reduce organic groups on the surface of the carbon dots,
promoting lower luminescent intensity. Therefore, the best
synthesis conditions for preparing carbon dots must be
Table 2 Experimental conditions for obtaining carbon dots from nopal established.
The color emission of CDs depends on the particle size and
Temperature Time Concentration Concentration
Sample (1C) (h) (mg mL 1) input (mg mL 1) output distribution; therefore, they have a tunable color. For this
reason, the chromatic coordinates of the carbon dots are reported
1 200 2.5 25 0.75
2 200 2.5 100 1.70 in the present study in order to distinguish the color of emission
3 200 5 25 1.05 (Fig. 2). Only a blue color emission of CDs was observed by human
4 200 12 25 1.27 eyes when excited at 375 nm wavelength, however, CIE chromaticity
5 180 12 25 0.95
6 160 12 25 0.83 diagram revealed a difference in the color emission among the
samples (Fig. 3). Samples 2, 3, 4 and 5 exhibit very similar emission
color, indicating that these samples have similar particle
distributions. The chromatic coordinates of samples 1 and 6
had a slight difference in the emission color with respect to the
other samples; such a difference can be attributed to the carbon
dot concentrations, 0.75 and 0.83 mg mL 1, respectively. Sample 4
had the maximum emission intensity and its chromatic coordinates
were x = 0.165 and y = 0.172. Due to this result, sample 4 was used
for determining the genotoxic and cytotoxic potential.
Raman spectra of the carbon dots coming from guava,
cucumber, nopal, and avocado are shown in Fig. 4. In the
800–200 cm 1 region, two characteristic peaks corresponding
to the G and D bands are observed. The D band located around
1360 cm 1 is associated with vibrations of carbon atoms with
dangling bonds or glassy carbon; the G band is related to defects
and disordered carbon associated with the two-dimensional
hexagonal lattice of the graphite structure.50 Thus, the Raman
Fig. 2 Emission spectra and chromatic coordinates of CDs obtained from spectra indicate the co-existence of the amorphous and crystalline
nopal. structure of carbon.51 There are slight differences among the D

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Fig. 3 Chromatic coordinates of the CDs obtained from nopal.
Fig. 4 Raman spectra of the carbon dots from different plants.
band positions of the carbon dots as a result of the carbon source
composition; similar differences also were found for the G bands.
The intensity ratios of the D band and the G band of the
carbon dots were 1.1, 0.90, 0.89, and 0.83 for avocado, cucum-
ber, guava and nopal, respectively. When the intensity of the G
band is higher than the D band, the carbon dots contains
principally sp2 carbon with some sp3 hybrid carbon.52 Thus, the
nopal carbon dots may contain a high percentage of graphite
and a low percentage of carbon atoms with dangling bonds.
3.2 FT-IR and UV-vis analyses of the CDs
The functional groups of the studied CDs were identified by
FT-IR spectroscopy. The infrared and UV spectra were generally
quite similar between the CDs whatever the carbon source; for
this reason, we show the infrared and UV spectra of the CDs
obtained from nopal at 25 mg mL 1, 12 h and 200 1C (Fig. 5a).
The absorption bands shown in Fig. 5a reveal that the bonds
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Fig. 5 (a) Infrared and (b) UV spectrum of the carbon dots (synthesized at
200 1C, 12 hours and 25 mg of nopal).
mainly consist of carbon, oxygen and hydrogen atoms. Firstly,
the broad band ranging from 3100–3500 cm 1 was related to
the stretching vibration of hydroxyl groups (–OH) coming from
water adsorbed on the CDs,47,53 while the C–H asymmetric
vibrations due to the presence of the alkyl groups were located
at 2905 cm 1.53 The absorption bands around 1550 and
1390 cm 1 are assigned to stretching vibrations of CQO and
CQC groups, suggesting the presence of carboxyl groups.
Finally, the absorption band located at 1070 cm 1 corresponds
to the symmetric stretching vibration for C–O–C.47,54
The UV spectrum of the CDs (Fig. 5b) exhibits two absorption
bands located around 250 and 315 nm corresponding to n–p*
and p–p*, which are ascribed to CQO and to a conjugated C,
respectively.48,55
3.3 High resolution transmission electron microscopy
analyses of the CDs
According to our results, sample 4 and sample 6 had the highest
and the lowest emission intensity, respectively (see Table 2). These
samples were characterized by HR-TEM in order to determine
their structure and particle size distribution. Fig. 6 reveals a low
magnification HR-TEM analysis of the CD samples as well as the
particle size distribution measured by image analyzer software.
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Fig. 6 Particle size distribution of the carbon dots: (a) sample 6 (synthe-
sized at 160 1C, 12 h and 25 mg of nopal), and (b) sample 4 (synthesized at
200 1C, 12 h and 25 mg of nopal).
The particle sizes of sample 6, which corresponded to 25 mg of
nopal CDs prepared at 160 1C and 12 h, were statistically
estimated to be 4.63–8.17 nm, that is, much higher than the
25 mg of nopal CDs prepared at 200 1C and 12 h, which were
computed to be 2.3–5.77 nm. Therefore, the highest temperature
of the reaction seemed to reduce the particle size of the CDs, since
the reaction time and nopal concentration were kept constant in
both syntheses. This observation agrees with the results of Liu
et al.;56 they found that an increased reaction temperature leads to
a decrease in particle size. Their results showed that at temperatures
of 150, 180 and 200 1C the diameter ranges of the carbon dots were
18–22, 3–5 and 2–3 nm, respectively.
The HR-TEM images of the CDs are shown in Fig. 7. Sample
6 and sample 4 revealed well crystallized nanoparticles since
well-resolved lattice fringes for both samples can be seen. The
measured lattice spacing of 2.46 Å and 3.15 Å corresponds to
the (100) and (002) crystallographic planes, respectively. These
distances of the indexed planes match very well with the close-
packed hexagonal structure of carbon. Zheng et al.42 also
reported a similar lattice spacing (2.46 Å) for the (100) plane
for highly luminescent carbon dots obtained by a vitamin-
based small organic molecule with a benzene ring structure
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Fig. 7 HR-TEM image of the CDs: (a) sample 6 (synthesized at 160 1C,
12 hours and 25 mg of nopal), and (b) sample 4 (synthesized at 200 1C,
12 hours and 25 mg of nopal).
with an average size of about 2.5 nm, while Ding et al.57
reported luminescent carbon dots with an average diameter
of about 4 nm. The latter research group obtained a lattice
spacing of 0.32 nm, a result that coincides with the lattice
spacing reported in the present work (3.15 Å) for the (002)
plane. Nevertheless, the lattice spacings for the (100) and (002)
planes reported in ICDD card #00-023-0064 for bulk graphite
are 2.13 and 3.35, respectively. Thus, the nanosize scale of the
carbon dots might affect the lattice spacing.
3.4 Genotoxic and cytotoxic analysis of the nopal CDs
Regarding the genotoxic potential of the CDs our results are
shown in Fig. 8. As expected, a low and constant level of MNPE
in the control animals was observed along the whole assay, as
well as highly significant damage induced by the mutagen
(doxorubicin) from 24 to 96 h, although with a certain micro-
nuclei formation decrease in the last two days. This last result
was congruent with that usually determined when a single
administration is given, that is, the stronger damage is
generally found at 48 h followed by a certain decrease related
to the detoxification and DNA repair processes, as well as to the
duration of erythropoiesis. Doxorubicin is an anticancer drug
that acts against a number of malignancies including solid
tumors, leukemia, and lymphoma. The drug is able to induce
free radicals and interacts with DNA by intercalation. These
actions explain its well-known genic and chromosome damage,
a capacity that has led to the use of the compound as a positive
control in genotoxicity assays since the 1990s.58,59 With respect
to the effect of the CDs, it was interesting to observe genotoxicity
with all tested doses, the two low ones being the less genotoxic,
and statistically not significant at 96 h post exposure. We also
found that 48 h post exposure corresponded to the highest
micronuclei elevation with the three high tested doses; at this

NJC
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Fig. 8 Micronuclei polychromatic erythrocytes (MNPE) induced by the
CDs in mouse peripheral blood. Each bar represents the mean  standard
error (SEM) obtained in 6 mice per group. 1000 polychromatic erythro-
cytes per mouse. * statistically significant difference with respect to the
control group value. ANOVA and post hoc comparison with the Student–
Newman–Keuls test (P o 0.05).
time-point an increase of more than four times was determined
with respect to the control level.
Fig. 9 presents the results obtained with respect to bone
marrow cytotoxicity. In this case, no effect was determined over
bone marrow proliferation in the control mice, contrary to a
significant decrease in the number of polychromatic erythro-
cytes induced by doxorubicin from 24 to 96 h of the assay, a
result which indicated a cytotoxic effect reflected in lesser produc-
tion of young erythrocytes. The CDs also induced a proliferation
decrease along the assay, particularly 48 h after exposure. At this
time, the mean inhibitory effect induced by the three highest
doses of CDs was 34.6% compared with the value determined in
Fig. 9 Mouse bone marrow cytotoxic effect induced by the CDs measured
by the proportion of polychromatic erythrocytes (PE) with respect to the
amount of erythrocytes. Each bar represents the mean  standard error (SEM)
obtained in 6 mice per group. 1000 polychromatic erythrocytes per mouse.
* statistically significant difference with respect to the control group value.
ANOVA and post hoc comparison with the Student–Newman–Keuls test
(P o 0.05).
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the control mice. Thus, our present geno/cytotoxicity results
revealed that the CDs assayed in the present conditions were able
to damage DNA and inhibit bone marrow cell proliferation,
particularly with the last three high tested doses.
Studies on geno/cytotoxicity establish the capacity of CDs to
damage genetic material and affect cell viability, which are
relevant determinations for biomedical purposes, where the
better CD concentrations should be coupled with low geno and
cytotoxic effects. Moreover, these data in normal cells may
allow one to adjust the adequate range of CD concentration
in carriers of biomolecules or to test the potential selectivity of
the nanoparticles in cancerous cells. In this context, Pan
et al.,60 for example, constructed immunosensors synthetized
from two CdTe quantum dots and examined their in vitro
cytotoxicity, concluding that one of them (CdTe:SiO2) exhibited
lower cytotoxicity and higher electrochemiluminescence and,
therefore, it was appropriate for their purpose.
The observed micronuclei represent chromosomal fragments,
or whole chromosomes abnormally segregated into daughter cells
during the process of mitosis. Such a determination corresponds
to one of the most used genotoxicity tests because micronuclei
can be obtained in most dividing cells independently of the
organism’s karyotype, they can be easily identified and they
provide accurate data; moreover, the background frequencies of
micronucleated cells are usually stable.39
Studies to determine CDs’ genotoxic capacity are increasing
in light of the potential biomedical application of such nano-
particles. This type of studies has been performed mainly in a
number of cultivated cells and, to a minor extent, using in vivo
assays. Investigations on the matter have included single and
multi walled carbon nanotubes by examining the DNA strand
breakage potential, as well as their effect in micronuclei and
chromosomal aberration induction, among other endpoints.61
The obtained results have been variable, depending on the
specific source to obtain the nanoparticle and on its physical
characteristics, as well as on the used experimental conditions
and the applied test, a situation that confirms the relevance of
genotoxic evaluation of each newly constructed nanoparticle.
Additionally, it was reported that CDs prepared using an
improved nitric oxidation method were found to be safe according
to various toxicological assays, including the micronucleus and
the Ames test27 results, which suggests that modifications to the
experimental conditions may change the toxicological response.
When CDs are obtained by the hydrothermal method, first, an
extraction process occurs, therefore secondary metabolites
belonging to nopal are obtained. At a certain temperature the
carbonization process occurs. However, if the carbonization is
incomplete, secondary metabolites could remain and may be
involved in cytotoxicity or genotoxicity. According to the literature,
the phytochemical screening of Mexican nopal extraction using
water indicates phenols, flavonoids, condensed tannins, hydro-
lyzable tannins, saponins and phytosterols,62 while other researchers
have also reported alkaloids. Most studies have focused on the
genotoxicity and cytotoxicity of functionalized CDs or in the presence
of capping agents, but these studies hardly ever describe the surface
behavior of CDs in nature. It is necessary to know the CD behavior
New J. Chem.

Paper
resulting from natural sources. In this context, the cytotoxicity and
genotoxicity of the synthesised carbon dots were evaluated. The
detection of tannins, phenols, saponins, alkaloids and flavonoids
was negative, suggesting that the secondary metabolites were
carbonized. Thus, the genotoxicity and cytotoxicity can be attributed
to the carbon dot surface generated by the synthesis conditions and
the carbon source. Therefore, we are presently modifying the nopal
extraction process, hydrothermal reaction temperature and time,
and the CD separation process. With these changes, a reduction in
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genotoxicity was observed in a preliminary assay.
With respect to the underlying mechanisms to explain the
reported DNA damage, oxidative stress has been mentioned as
the main activity involved, together with alterations in DNA
repair, chromosome breakage, aneuploidy, or even deforma-
tion at the end of DNA base pairs, which also promote geno-
toxicity; however, interestingly no gene mutations (at least in
bacterial systems) have been detected.63
In our assay, inhibition of bone marrow proliferation was
observed with doxorubicin, as well as with various doses of the
tested CDs, mainly with the three highest doses at 48 h post-
exposure. The cytotoxicity of nanoparticles has been mainly
evaluated in cultivated cells through the use of various
methods, such as the trypan blue dye exclusion assay, and
others colorimetric based assays using tetrazolium salts.64,65
The processes involved in such studies are known to include
activation of molecules that results in apoptosis, necrosis
or autophagy;66,67 however, investigations analyzing global
physiological effects in whole animals have also been performed,
for example in Xenopus laevis and in Caenorhabditis elegans.68,69
The geno/cytototoxic results obtained in the present assay
are useful as basic toxicological knowledge of the tested nano-
particles, but they can also be used to support future biomedical
studies. Confirmation of the presently obtained information with
other tests is always valuable; however, here we have established
high DNA damage potential with most CD tested doses, with the
exception of the first low dose, suggesting that at such a low
range the capability of these nanoparticles can be assayed to
transport biomarkers of disease, for imaging purposes or to
examine their effect of selectively destroying cancer cells, as has
already been described for colon cancer cells with disulfiram-
loaded charge switchable nanoparticles or for hepatic cancerous
cells with charge switchable diethylditiocarbamate loaded
nanoparticles.70,71
4. Conclusions
Photoluminescent CDs from Opuntia ficus-indica, Inga feuilleei,
Cucumis sativus, and Persea americana were obtained by the
hydrothermal method. In our study we confirmed that the
carbon dot concentration is a function of the reaction temperature
and time, and the concentration of the carbon source (nopal); these
parameters promote an ‘‘optimum concentration’’ of carbon dots
which maximizes the luminescent intensity response. The CDs
obtained from Opuntia ficus-indica presented the highest emission
intensity when compared to those from I. feuilleei C. sativus, and
New J. Chem. This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
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NJC
P. americana. We found that the nopal CDs presented blue
emission, confirmed by chromatic coordinates x = 0.157 and
y = 0.158, and that they crystallized in a hexagonal phase.
Moreover, a strong genotoxic capacity of the nopal CDs was
also demonstrated, measured with the micronucleus test in
mice, as well as a high cytotoxic effect according to the relation-
ship between the amount of polychromatic and normochromic
erythrocytes. These are data that should be useful in future
studies regarding applications in the biomedical field, particularly
at the lowest dose range, or as the basis to look for experimental
changes that may reduce the observed toxicity.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors gratefully acknowledge the CCAI-CENTER of
UTTEC for the use of facilities during the carbon dot syntheses
as well as CIITEC-IPN, CNMN-IPN and ENCB-IPN for the
characterization and biological studies.
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