Single and repeated dose toxicity of citric acid-based carbon dots and a derivative in mice

Article abstract of DOI:10.1039/c5ra18391j

Carbon dots (CDs) have recently emerged as a new class of fluorescent nanomaterials and are already competitive in many respects to conventional semiconductor quantum dots (QDs). Despite CDs remarkable advantages in bioimaging, biosensing and other biomedical applications, their biosafety is still unclear. In the present study, the systematic single and repeated dose toxicity and biodistribution of two citric acid-based probes (CDs and a derivative, Et-IPCA) were investigated in vivo. For the single dose toxicity studies, the lethal dose 50 (LD₅₀) of CDs was 391.615 mg kg^-1 and 357.771 mg kg^-1 for female and male mice, respectively, while Et-IPCA reached its saturation point in solution at 25 mg kg^-1 and did not lead to the death of any mice at that concentration. For the repeated dose toxicity studies, although there were temporary changes in hematological parameters suggesting an acute inflammatory response after seven doses, the data on the body weight, organ coefficients, hematological parameters, blood biochemistry, and organ histopathology at the end of the 90 day recovery period suggested that both the CDs and Et-IPCA had low toxicity over the 90 day period. These findings will be useful for the future development of CDs-based drug delivery systems and for the development of clinical applications of these systems.

Full text of DOI:10.1039/c5ra18391j

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PAPER
Single and repeated dose toxicity of citric acid-
based carbon dots and a derivative in mice†
Cite this: RSC Adv., 2015, 5, 91398
a
a
a
b
a
a
Xiao Zheng,‡ Dan Shao,‡ Jing Li, Yubin Song, Yujing Chen, Yue Pan,
b
b
ac
Shoujun Zhu, Bai Yang* and Li Chen*
Carbon dots (CDs) have recently emerged as a new class of fluorescent nanomaterials and are already
competitive in many respects to conventional semiconductor quantum dots (QDs). Despite CDs
remarkable advantages in bioimaging, biosensing and other biomedical applications, their biosafety is still
unclear. In the present study, the systematic single and repeated dose toxicity and biodistribution of two
citric acid-based probes (CDs and a derivative, Et-IPCA) were investigated in vivo. For the single dose
ꢀ1
ꢀ1
toxicity studies, the lethal dose 50 (LD50) of CDs was 391.615 mg kg and 357.771 mg kg for female
ꢀ1
and male mice, respectively, while Et-IPCA reached its saturation point in solution at 25 mg kg and did
not lead to the death of any mice at that concentration. For the repeated dose toxicity studies, although
there were temporary changes in hematological parameters suggesting an acute inflammatory response
after seven doses, the data on the body weight, organ coefficients, hematological parameters, blood
biochemistry, and organ histopathology at the end of the 90 day recovery period suggested that both
the CDs and Et-IPCA had low toxicity over the 90 day period. These findings will be useful for the future
development of CDs-based drug delivery systems and for the development of clinical applications of
these systems.
Received 9th September 2015
Accepted 19th October 2015
DOI: 10.1039/c5ra18391j
www.rsc.org/advances
because they are heavy-metal-free. Hence, much research using
1
. Introduction
12,13
14
CDs for optical bioimaging
and drug delivery has been
The importance of uorescent probes in biomedical research carried out. On the other hand, the biocompatibility of CDs
15
and practice is rapidly increasing with the rapid developments remains a key reason for restricting their further use in vivo.
in uorescence microscopy, laser technologies, and nanotech-
The toxicity of CDs has already been reported, and the results
nology. Since the discovery of uorescent fragments in a batch showed that it would be safe to use these kinds of CDs as bio-
1
2,3
16–18
of single-wall nanotubes in 2004, carbon dots (CDs) of logical probes for optical bioimaging.
Zhang's group has
different intrinsic structures, including graphene quantum dots studied the synthesis, characterization, in vitro toxicity and in
4
–6
7–9
(
(
GQDs),
PDs),
carbon nanodots (CNDs)
have been prepared and characterized. These uo- graphene oxide (GO). They found that the CDs showed no
and polymer dots vivo biodistribution and toxicity of a CDs sample obtained from
1
0,11
17
rescent carbon-based materials have drawn increasing atten- obvious inuence on mice owing to their small size, while GO
tion for their exceptional advantages, such as high optical appeared toxic, and even caused death in mice. CDs possess no
absorptivity, chemical stability, biocompatibility, and low obvious in vitro or in vivo toxicity, even aer multiple doses.
toxicity, which distinguish them from traditional uorescent Apart from that study, Cui's group has also successfully
materials and make them promising candidates for numerous prepared CDs with good stability, high dispersibility, and water
18
applications. Compared to traditional quantum dots (QDs), solubility. They have demonstrated that no signicant toxic
CDs have shown greater potential for biological applications effects, and no abnormalities or lesions were observed in the
organs of the animals at all studied CDs doses. However, more
efforts are still required to fully assess the toxicity of CDs
considering that there exist so many carbon-based materials
which possess the potential to be prepared into CDs. Accord-
ingly, an improved understanding of the toxicity of various CDs
a
Department of Pharmacology, Nanomedicine Engineering Laboratory of Jilin Province,
College of Basic Medical Sciences, Jilin University, Changchun 130021, China. E-mail:
chenl@jlu.edu.cn
b
State Key Laboratory of Supramolecular Structure and Materials, College of
is essential, and long-term toxicological and pharmacokinetic
Chemistry, Jilin University, Changchun, 130012, P. R. China. E-mail: byangchem@
jlu.edu.cn
investigations involving the degradation, excretion, persistence
and immune response associated with CDs will ultimately be
c
School of Nursing, Jilin University, Changchun 130021, China
†
1
‡
Electronic supplementary information (ESI) available. See DOI: needed to design carbon-dot-based probes for clinical imaging
0.1039/c5ra18391j
and therapy.
These authors contributed equally.
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We have previously established a facile and high-output 5 mmol EDA (335 mL) were dissolved in ultrapure water (10 mL).
strategy for the fabrication of CDs using citric acid and ethyl- Then, the solution was transferred to a poly(tetrauoroethylene)
9
ꢁ
enediamine using a hydrothermal method, and obtained (Teon)-lined autoclave (25 mL) and heated at 200 C for 10 h.
polymer-like CDs. On the basis of this strategy, a distinct bright Aer the reaction, the reactors were allowed to cool naturally to
blue uorophore (imidazo[1,2-a]pyridine-7-carboxylicacid, 1,2,3,5- room temperature. The CDs solution was then evaporated to
19
tetrahydro-5-oxo-, IPCA) was extracted. It was proven to be an obtain the CDs powder. Et-IPCA was synthesized by hydro-
important component in CDs and the photoluminescence center thermal treatment and was puried by column chromatography
19
for the molecular state. However, pure IPCA is not stable in as reported previously. A total of 1 mmol CA (0.1921 g) and 1
diluted aqueous solutions, in which light and/or oxygen can affect mmol Et-EDA (67 mL) were dissolved in ultrapure water (10 mL),
its photoluminescence (PL) behavior. We thus improved the then, the solution was transferred to a poly(tetrauoroethylene)
ꢁ
starting materials using N-ethylethane-1,2-diamine (Et-EDA) (Teon)-lined autoclave (25 mL) and heated at 140 C for 10 h.
instead of EDA and obtained 1-ethyl-5-oxo-1,2,3,5-tetrahy- Aer reaction, the reactors were cooled to room temperature by
19
droimidazo-[1,2-a]pyridine-7-carboxylic acid (Et-IPCA). CDs are water or were allowed to cool naturally to obtain a uorescent
nano-material, while Et-IPCA is more like an organic dye. Et-CDs solution. Et-IPCA was extracted from the solution by
However, both of them are citric acid-based probes and share the column chromatography using methanol as the developing
advantages of facile synthesis, water solubility and a high solvent.
quantum yield. Compared with CDs, Et-IPCA seems to be advan-
tageous because of its fully resolved chemical structure, resistance
to photo-bleaching and higher quantum yield. Although these two
2
.3 Characterization of the CDs and Et-IPCA
Fluorescence spectroscopy was performed using a Shimadzu
RF-5301 PC spectrophotometer. UV-vis absorption spectra were
obtained using a Shimadzu 3100 UV-vis spectrophotometer.
Nanosecond uorescence lifetime experiments were performed
on a uorescence spectrometer. (Edinburgh Instrument, FLS
uorescent probes have shown great potential in bioimaging,
assessments of their biosafety have not been carried out.
Herein, we systematically studied the single- and repeated-
dose toxicity of these citric acid–base probes (CDs and
Et-IPCA) in ICR mice. The CDs and Et-IPCA were synthesized via
a simple, scalable method, then their cytotoxicity to four
different cell lines (HepG2, L02, MCF-7 and HUVEC) was
determined by the SRB assay, and hemolysis was also assessed.
To mimic clinical drug administration and to more practically
study the effects of carbon-based probes in mice, both single-
and repeated-dose toxicity testing were carried out using studies
designed according to State Food and Drug Administration's
980). High-resolution transmission electron microscopy
(HRTEM) was performed using a JEM-2100F instrument. NMR
spectra were obtained on an AVANCEIII500 (500 MHz) from
Bruker. Dimethyl sulfoxide-d6 (with 0.03% TMS) was chosen as
the solvent for the samples.
2.4 Cell culture and cytotoxicity assessment
(SFDA) acute and chronic toxicity test technical guidelines for The human hepatocellular carcinoma cell line (HepG2), human
chemical drugs (guidelines). In the repeated-dose toxicity test, hepatic embryo cell line (HL-7702; L02), human breast cancer
the body weight, hematological parameters, blood biochem- cell line (MCF-7), and human umbilical vein endothelial cell-
ꢁ
istry, and organ histology were determined at 1, 7, 30, and s(HUVEC) were maintained at 37 C under 5% CO in RPMI-
2
9
0 days aer beginning administration. Moreover, the bio- 1640 supplemented with 10% (v/v) heat-inactivated fetal
ꢀ
1
distribution of these two probes was examined as well. The bovine serum, penicillin (100 U mL ), and streptomycin (100
results suggest that both CDs and Et-IPCA can serve as optical mg mL ). The in vitro cytotoxicity of CDs and Et-IPCA in the L02,
ꢀ
1
probes for specic clinical applications.
HepG2, MCF-7 and HUVEC cells were assessed using traditional
SRB assays. The sulforhodamine B (SRB) assay is routinely used
for cytotoxicity determination, based on the measurement of
the live cell protein content. In general, cells were seeded into
96-well plates and then treated with the indicated concentra-
2. Experimental section
2.1 Materials
ꢀ
1
tions (0, 3.125, 6.25, 12.5, 25, 50, 100, 250, and 500 mg mL ) of
CDs or Et-IPCA for 72 h. Aerwards, 100 mL of 20% trichloro-
acetic acid (TCA) was added to the culture medium in each well,
Citric acid (CA) was purchased from Aladdin (citric acid anhy-
drous, 99.5%), ethylenediamine (EDA) and N-ethylethane-1,2-
diamine (Et-EDA) were purchased from Alfa Aesar (99%). Sul-
forhodamine B was purchased from Sigma-Aldrich. RPMI-1640
medium was obtained from GIBCO. Fetal bovine serum (FBS),
penicillin and streptomycin were from the Beyotime Institute of
Biotechnology (Jiangsu, China). All reagents were commercially
available products with analytical grade purity and were used
without further purication.
ꢁ
and the plates were stored at 4 C for 1 h. Aer that, the plates
were washed three times with a gentle stream of deionized
water, and the excess water was tapped out onto paper towels. A
blow dryer was used to completely dry the plates. Then, 100 mL
of 0.4% SRB, which included 1% acetic acid, was added to each
well, and the plates were incubated for 30 minutes at room
temperature. Aer icking the SRB off and washing the plates
with 1% acetic acid, the plates were dried and 150 mL of 10 mM
Tris base solution was added to each well. The samples were
2.2 Synthesis of CDs and Et-IPCA
CDs were prepared by a bottom-up carbonization method as shaken for 5 minutes. The absorbance was read by a microplate
9
reported previously. Typically, 5 mmol CA (0.9605 g) and reader at 570 nm. The survival rate of the cells was expressed as
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the A/B 100%, where A was the absorbance value from the
experimental cells and B was that from the control cells.
One hundred ICR mice were divided randomly into ve
groups, a control group and four experimental groups, with ten
male and ten female mice in each group. The doses used in the
ꢀ
1
ꢀ1
ꢀ1
LD50 experiment were 250 mg kg , 320 mg kg , 400 mg kg
2.5 Hemolytic testing
ꢀ1
and 500 mg kg . All mice were fed adaptively for seven days,
To study the hemolytic characteristics of CDs and Et-IPCA, then were treated with different concentrations of CDs saline
a classic hemolytic test was conducted. First, 50 mL of rabbit solutions via a tail vein and were observed for 14 days. Mortality
blood in a glass ask was stirred with a cotton swab to remove was recorded, and all survival mice were sacriced. The value of

brous proteins. Then, 100 mL of saline was added. The the LD50 was calculated using the Origin 6.0 soware program.
mixture was centrifuged at 1500 rpm for 15 min, and the sedi-
ment was reserved. This procedure was repeated twice until the
supernatant was clear. Aer that, we generated 2 mL of red
blood cell (RBC) sediment, which was red blood cells (RBCs)
mixed with 100 mL of saline to obtain a 2% RBC suspension.
The CDs and Et-IPCA were dissolved to different concentrations
2
.7 Repeated-dose toxicity testing
For the subsequent repeated-dose toxicity study, 160 male ICR
mice were randomly divided into groups that received CDs
(60 mice), Et-IPCA (60 mice) or the control (40 mice). The mice
ꢀ
1
were injected with CDs (100 mg kg , by tail vein), Et-IPCA
ꢀ
1
(12.5, 25, 50, 100, 250, and 500 mg mL ) with saline. Then,
ꢀ
1
(25 mg kg , by intraperitoneal injection) or saline once a day
2.5 mL of the above solutions and 2.5 mL of RBC suspension
for one week. Then, all mice were weighed once a week, and
1
9
observed and recorded carefully throughout the entire study. At
the end of the study, all animals were sacriced.
were mixed in different glass test tubes. We used 2.5 mL of
saline and deionized water with 2.5 mL of RBC suspension as
negative and positive controls, respectively. The mixtures were
gently shaken up and then allowed to settle for 3 or 24 hours at
room temperature. Finally, the mixtures were centrifuged for
0 mice from each group were sacriced on days 1, 7, 30 and
0 days aer injection. The symptoms and mortality were
2
min at 4000 rpm, and the absorbances of the upper clear
2.8 Hematological analysis and blood biochemical assay
solutions were recorded by a microplate reader at 540 nm. The
percentage of hemolysis in each of the samples was calculated Blood drawn from the eyeball was collected for blood tests. To
by dividing the difference in absorbance between the sample reveal any potential toxic effects of CDs and Et-IPCA on the
and the negative control by the difference in absorbance treated mice, blood biochemistry and hematological analyses
between the positive and negative controls.
were carried out. Important markers, including the numbers of
white blood cells and red blood cells, the hemoglobin level,
mean corpuscular volume, mean corpuscular hemoglobin,
mean corpuscular hemoglobin concentration, and platelet
2.6 Animals and single-dose toxicity testing
All animal experimental protocols were approved by the Ethics count for the hematological assessment, and the alanine
Committee for the Use of Experimental Animals of Jilin aminotransferase (ALT), aspartate aminotransferase (AST),
University. Both sexes of ICR mice (aged 6–8 weeks) were used in alkaline phosphatase (ALP), total bilirubin (TB), albumin (ALB),
the experiments. Ten of each sex of mice were housed in blood urea nitrogen (BUN), serum creatinine (CRE), total
stainless steel cages containing sterile paddy husks as bedding cholesterol (TC) and total triglycerides (TG) for the blood
in ventilated animal rooms. The mice were acclimated to the biochemistry studies were selected.
ꢁ
controlled environment (temperature: 25 C; humidity: 60%
and light: 12 h light/dark cycle) with free access to water and
a commercial laboratory complete chow.
2
.9 Organ coefficients and histopathological examinations
The tissues and organs, such as the liver, spleen, kidney, heart,
lung, and brain were excised and accurately weighed. The
coefficients of the liver, spleen, kidneys, heart, lungs, and brain
to body weight were calculated as the ratio of the tissues (wet
weight, mg) to the body weight (g). Then, these tissues recovered
from the necropsy were xed in 10% formalin, embedded in
paraffin, sectioned, and stained with hematoxylin and eosin
The single-dose toxicity testing was performed before the
repeated-dose toxicity testing to investigate the preliminary
toxicity proles of CDs and Et-IPCA, as well as to determine the
doses that would be used in the latter studies. In general, the
median lethal dose (LD ) was calculated by an improved Bliss
5
0
method. Several groups of ICR mice were used in a pre-test to
determine the minimum dose (D ) and maximal dose (D ) of
n
m
(H&E) and examined under a digital microscope.
CDs and Et-IPCA. Intravenous injections of CDs and Et-IPCA
solutions, which were prepared in physiological saline, were
2.10 Biodistribution of the CDs and Et-IPCA
n m
conducted through a mouse tail vein. The D and D for CDs
ꢀ1
ꢀ1
were 250 mg kg and 500 mg kg , respectively. With regard to For the in vivo biodistribution studies, 30 male ICR mice were
Et-IPCA, it reached its saturation for solubility in saline at 25 mg randomly into a CDs group, Et-IPCA group and control group
ꢀ
1
ꢀ1
ꢀ1
kg , and all mice survived aer treatment at that dose. Based and were injected with CDs (25 mg kg ), Et-IPCA (25 mg kg
)
on these ndings, combined with those of previous results, or saline through a tail vein. Then, ve mice from each group
we concluded that Et-IPCA was safe to use for single-dose were sacriced at three hours and 24 hours aer injection.
treatment, and the single dose toxicity of CDs was further Approximately 1.0 mL of blood from each mouse was collected
investigated.
and centrifuged at 3500 rpm for 10 minutes to collect serum.
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Then, the serum was diluted four-fold with saline. Major CDs can be conrmed by TEM, and Fig. 1b showed that the CDs
organs, including the liver, spleen, lungs, heart, kidneys and had a uniform dispersion without any aggregation, with the
brain, from these mice were harvested and stored in 3 mL of diameter of particles being between 1–5 nm. The structure of Et-
1
saline. These organs were homogenized, and the tissue IPCA can be conrmed from the H-NMR spectra (Fig. 1c).
homogenates were centrifuged at 3500 rpm for 10 minutes. The Notably, both the CDs and Et-IPCA solutions could be stored at
supernatants were collected and examined on a spectro- room temperature for as long as 90 days without the formation

uorophotometer with a 360 nm excitation wavelength, then of any precipitates or a loss of uorescence (Fig. S1, ESI†).
the photoluminescence (PL) intensity of the emission spectra at Collectively, these ndings indicate that the prepared CDs and
450 nm were recorded.
Et-IPCA have good biocompatibility and photostability.
2.11 Statistical analysis
3.2 Cellular toxicity and hemolytic testing of CDs and Et-
The data were expressed as the means ꢂ SD. The statistical IPCA
signicance of the data was compared by Student's t-test. An
The rst set of experiments in this study compared the toxicity
of CDs and Et-IPCA in vitro. The human hepatocellular carci-
noma cell line (HepG2), human hepatic embryo cell line
analysis of variance (ANOVA) was used to analyze the differ-
ences among the different groups. The level of statistical
signicance for all tests was set at P < 0.05.
(HL-7702), human breast cancer cell line (MCF-7), and human
umbilical vein endothelial cells (HUVEC) were chosen for these
investigations and were cultivated in the presence of increasing
3
. Results and discussion
ꢀ
1
concentrations of CDs and Et-IPCA (1.5625 mg mL to 500 mg
3.1 Synthesis and characterization of CDs and Et-IPCA
ꢀ1
mL ). The cell viability was measured aer a 72 h incubation
using the sulforhodamine B(SRB) assay. As shown in Fig. 2, no
signicant reduction in cell viability was observed aer the 72 h
incubation with either CDs or Et-IPCA, even when the concen-
Our group previously reported a facile and high-output strategy
for the fabrication of high-quantum-yield CDs from citric acid
(CA) and ethylenediamine (EDA), as well the discovery of a type
9,19
of bright blue uorophore (Et-IPCA) derived from CDs. These
highly photoluminescent probes have drawn increasing atten-
tion for their potential applications in multicolor patterning,
biosensors, and cell imaging due to their advantages over other
ꢀ1
tration of the CDs was 500 mg mL , demonstrating that these
citric acid-based probes were not obviously toxic in vitro. Similar
phenomena were found in the case of PC12 and RSC96 cells in
20
our previous study, indicating that these citric acid-based

uorophores. As shown in Fig. 1a, the Et-IPCA showed a strong
probes are not obviously toxic to cultured cells.
blue PL in an aqueous solution, and its absorption peaks were
at 242 nm and 360 nm, which is similar to that of CDs. However,
the Et-IPCA exhibited a stronger uorescence intensity and
better photostability than CDs under excitation at 360 nm,
indicating that this molecule has stable optical properties that
may be useful in bioimaging applications. The appearance of
The hemolytic test is considered to be a supplementary test
for the assessment of cytotoxicity. It is used to evaluate whether
erythrocytes will dissolve and release hemoglobin aer direct
Fig. 1 Characterization of the CDs and Et-IPCA. (a) The UV-vis Fig. 2 The biocompatibilities of CDs and Et-IPCA. (a–d) The relative
absorption spectra and fluorescence emission spectra of CDs and Et- viabilities of HepG2 (a), L02 (b), MCF-7 (c) and HUVEC (d) cells after
IPCA. (b) High-resolution transmission electron microscope (HRTEM) incubation with CDs and Et-IPCA at various concentrations for 72 h.
1
images of the CDs and Et-IPCA. Scale bars are 20 nm. (c) H-NMR of Each bar represents the mean ꢂ SD of three independent experiments.
Et-IPCA.
(e) The results of the hemolysis assay of CDs and Et-IPCA.
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contact with the nanomaterials in the blood. It also can provide these studies have been conicting. In our study, the single-
a sensitive measure of the inuence of a nanomaterial on dose toxicity test was designed according to the SFDA's guide-
erythrocytes, and plays an important role in evaluations of lines. Although some criteria were changed to address the
21
biosafety. In the present study, the sedimentation of blood unique properties of CDs and Et-IPCA, this experimental design
cells was observed for both of the uorescent probes and for the can still be considered superior due to its use of a standard
normal saline group (Fig. S2, ESI†). However, the nal hemo- procedure and the discovery of the LD50
lysis rate of both the CDs and Et-IPCA leaching solutions was As was mentioned before, the results of the single-dose
less than 5%, even when the concentration of Et-IPCA reached toxicity testing of CDs differed between female and male
.
ꢀ1
5
00 mg mL , as shown in Fig. 2e. The high content of Et-IPCA in mice. Similar phenomena have been reported for many chem-
solution may cause a change in the osmotic pressure, which icals and traditional QDs. The reasons for this difference might
might be the reason for the hemolysis. However, this was not be rather complicated, including physical factors, differences in
a concern for further animal experiments because the nal enzyme systems and hormones. Aer considering this result,
concentrations of both the CDs and Et-IPCA would be relatively together with the results regarding the biodistribution and
16
23
low in vivo. Taken together, these results further demonstrated Liu's and Maysinger's reports, we assumed that physical
the good biocompatibility of these citric acid-based probes.
factors were likely play an important role because nanoparticles
of such a small diameter can be quickly excreted from the body,
which means that their half-life in vivo is relatively short, indi-
cating that they are unlikely to interact much with organs.
However, further testing is required to verify this assumption.
3.3 Single-dose toxicity of CDs and Et-IPCA
Because the results of the SRB assay and hemolytic test in vitro
showed that the CDs and Et-IPCA had acceptable biocompati-
bility, toxicity testing was carried out. No further experiments
were performed using Et-IPCA because neither death nor
abnormal behaviors were observed at its saturated concentra-
3.4 Repeated-dose toxicity of CDs and Et-IPCA
To further analyze the toxicity of repeated administration, mice
were dosed at different levels every day for seven days and were
observed for up to 90 days (Table 2, (a)). During the entire
experimental period, neither death nor abnormal behavior was
observed. To study the in vivo toxicity of CDs and Et-IPCA,
a series of experiments were conducted. First, to assess their
overall effects, the mice were weighed and then sacriced. Their
major organs, including the brain, liver, spleen, kidneys, heart
and lungs were collected and weighed. An organ coefficient,
which was the ratio of the organ weight to the body weight, was
calculated. No remarkable changes were found in body weight
in any of the groups, as shown in Fig. 3. Nevertheless, as is
shown in Table 2, (b), signicant changes could be found in
certain organs in the experimental groups at each time point,
indicating the possibility that there were injuries.
ꢀ1
tion in saline (25 mg kg ). As shown in Table 1, the mortality
ꢀ1
ꢀ1
rates following exposure to 320 mg kg and 400 mg kg were
different between female and male mice, indicating that the
single-dose toxicity of CDs is gender-related. The surviving mice
returned to normal within six hours, and their behaviors
showed no abnormalities compared with the control group
during the next 14 days, suggesting that CDs could be rapidly
eliminated in vivo. The LD50 of CDs calculated by linear inter-
ꢀ1
ꢀ1
polation were 391.62 mg kg and 357.77 mg kg for female
and male mice, respectively. It was concluded that male mice
are more sensitive to the higher concentrations of the CDs
solution than female mice. Based on this conclusion, we
believed that male mice would be a more suitable model for the
subsequent repeated-dose toxicity test. Single-dose toxicity
testing is always considered the rst step in toxicological safety
evaluations, and the LD serves as a key indicator of the toxicity
The immune system might have been stimulated by the
treatment because the size of CDs in vivo is similar to that of
viruses and large proteins, and this may have led to changes in
5
0
22
of an agent. Moreover, it is an important reference value to use
when designing repeated-dose toxicity tests. However, impor-
tant as it is, there has been little systematic research performed
with regard to the single-dose toxicity of CDs, and the results of
24
hematological factors. Accordingly, blood biochemistry and
hematological analyses were performed. As shown in Fig. 4 and
5, almost all of the parameters examined in the probe-treated
groups appear to be normal compared with the control group
and were within the normal ranges. It is notable that the white
blood cell count (WBC) (shown in Fig. 4a) suggested that both
CDs and Et-IPCA could induce inammatory responses, while
the effect of CDs lasted longer than that of Et-IPCA, for more
than 30 days. The changes in the red blood cell count (RBC) and
platelet count (PLT) shown in Fig. 4b, c and h supported this
conclusion.
The levels of alkaline phosphatase (ALP), a blood biochem-
istry factor reecting the health of the liver and bone, showed
signicant differences in the CDs and Et-IPCA groups compared
to the control in rst seven days aer treatment. However, these
differences were randomly distributed and do not appear to be
evidence of pathological changes in these tissues. The tissues
Table 1 The mortalities in the single-dose toxicity test of the different
concentrations of CDs and the LD50 values in female and male ICR
mice
ꢀ1
ꢀ1
Gender Dosages (mg kg
)
Death Mortality (%) LD50 (mg kg )
Female 250
0
0
391.615
3
4
5
20
00
00
1
5
10
0
10
50
100
0
Male
250
357.771
3
4
5
20
00
00
3
7
10
30
70
100
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Table 2 (a) The experimental design of the repeated-dose toxicity study. (b) The organ coefficients of male ICR mice at 1, 7, 30 and 90 days. Each
bar represents the mean ꢂ SD, n ¼ 10. P* < 0.05 vs. control group
(a)
(b)
Days aer
Body weight Liver coefficient Spleen coefficient Kidney coefficient Heart coefficient Lung coefficient Brain coefficient
treatment
Group (g)
(%)
(%)
(%)
(%)
(%)
(%)
1
Control 27.50 ꢂ 4.22 6.24 ꢂ 0.65
C-dots 27.97 ꢂ 3.13 6.43 ꢂ 0.68
0.58 ꢂ 0.07
0.67 ꢂ 0.09*
0.69 ꢂ 0.08*
0.96 ꢂ 0.09
0.87 ꢂ 0.10
0.95 ꢂ 0.07
0.71 ꢂ 0.08
0.60 ꢂ 0.06
0.63 ꢂ 0.06
0.86 ꢂ 0.12
0.90 ꢂ 0.14
0.94 ꢂ 0.11
1.53 ꢂ 0.22
1.44 ꢂ 0.18
1.50 ꢂ 0.21
Et-
27.99 ꢂ 3.88 6.03 ꢂ 0.63
IPCA
7
Control 30.03 ꢂ 2.25 5.18 ꢂ 0.58
0.53 ꢂ 0.08
0.56 ꢂ 0.11
0.53 ꢂ 0.07
0.72 ꢂ 0.08
0.91 ꢂ 0.11*
0.74 ꢂ 0.07
0.54 ꢂ 0.05
0.66 ꢂ 0.06*
0.62 ꢂ 0.06*
0.96 ꢂ 0.16
1.09 ꢂ 0.13
0.85 ꢂ 0.15
1.45 ꢂ 0.21
1.59 ꢂ 0.24
1.49 ꢂ 0.19
C-dots 28.82 ꢂ 1.83 6.19 ꢂ 0.69*
Et-
30.73 ꢂ 2.33 5.30 ꢂ 0.61
IPCA
3
0
0
Control 32.13 ꢂ 2.66 5.10 ꢂ 0.60
0.45 ꢂ 0.06
0.54 ꢂ 0.07
0.51 ꢂ 0.05
0.78 ꢂ 0.10
0.85 ꢂ 0.11
0.68 ꢂ 0.08*
0.50 ꢂ 0.06
0.63 ꢂ 0.06
0.61 ꢂ 0.07
0.76 ꢂ 0.14
0.85 ꢂ 0.12*
0.78 ꢂ 0.13
1.34 ꢂ 0.18
1.38 ꢂ 0.20
1.48 ꢂ 0.22*
C-dots 31.71 ꢂ 2.17 6.02 ꢂ 0.65
Et-
IPCA
32.18 ꢂ 2.25 5.26 ꢂ 0.63
9
Control 38.67 ꢂ 2.87 5.15 ꢂ 0.50
0.36 ꢂ 0.04
0.52 ꢂ 0.04
0.50 ꢂ 0.05
0.80 ꢂ 0.10
0.79 ꢂ 0.09
0.61 ꢂ 0.05*
0.47 ꢂ 0.04
0.60 ꢂ 0.06
0.61 ꢂ 0.06
0.66 ꢂ 0.09
0.78 ꢂ 0.08*
0.73 ꢂ 0.10
1.22 ꢂ 0.16
1.20 ꢂ 0.16
1.47 ꢂ 0.19*
C-dots 38.72 ꢂ 3.82 5.83 ꢂ 0.56*
Et-
IPCA
38.64 ꢂ 3.15 5.24 ꢂ 0.53
It should be noted that different methods of administration
were adopted in our repeated dose test, where the CDs were
injected via the tail vein while Et-IPCA was injected into the
peritoneal cavity. The reason we chose intraperitoneal admin-
istration for Et-PICA is that we concerned that there might be
a risk of sudden death if the Et-IPCA solution were injected
intravenously due to the volume of the Et-IPCA solution because
its solubility is much lower than that of CDs in saline (the
approximate injection volumes were 0.5 mL for Et-IPCA and 0.2
mL for CDs). Moreover, the difference in the absorption of these
materials between intravenous injection and intraperitoneal
injection is not that signicant. We considered that the method
Fig. 3 The body weights of male ICR mice in the control, CDs and Et-
IPCA groups at 90 days. Each bar represents the mean ꢂ SD, n ¼ 10. used in the current research on the in vivo toxicity of CDs, which
can be described as a one-time treatment and a subsequent long
observation period, was not sufficient to reveal all of the
were sliced and stained, then observed under a microscope to
potential effects of CDs. Furthermore, repeated-dose testing is
determine in detail whether there were any pathological
an essential part of the preclinical studies during drug devel-
changes in the CDs and Et-IPCA groups. It can be inferred from
Fig. 6 and 7 that there were no remarkable differences in any of
the major organs between the control and experimental groups.
Overall, the present data demonstrated that the CDs and Et-
IPCA at the concentrations used in this study did not lead to
signicant pathological changes based on the organ coefficient,
H/E staining and hematology analysis.
25
opment. A seven-day repeated treatment in animals is equiv-
alent to a single dose in human beings according to the
guideline, which served as the basis of our study design for our
repeated dose test. The time points we chose covered the whole
convalescent period and helped us to better understand the
biosafety of CDs and Et-IPCA.
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Fig. 6 Representative H&E-stained images of major organs, including
Fig. 4 The hematology data for male ICR mice treated with CDs at the liver, spleen, kidneys, heart, lungs and brain collected from the
ꢀ1
ꢀ1
ꢀ1
1
00 mg kg and Et-IPCA at 25 mg kg on days 1, 7, 30 and 90. Age- control untreated mice and CDs-injected (100 mg kg ) mice at
matched untreated mice were also sacrificed on days 1, 7, 30 and 90 as various time points post-injection. No noticeable abnormality or lesion
controls. (a–h) The time-course of the changes in white blood cells (a), was observed in any of the organs from the CDs-injected mice. All
red blood cells (b), hemoglobin (c), mean corpuscular volume (d), scale bars are 100 mm.
mean corpuscular hemoglobin (e), mean corpuscular hemoglobin
concentration (f), hematocrit (g), and platelets (h) in control mice, CDs-
treated mice and Et-IPCA-treated mice. Each bar represents the mean
ꢂ SD, n ¼ 10. P* < 0.05 vs. the control group. Gray bars indicate the
range of values obtained from healthy ICR mice.
Fig. 7 Representative H&E-stained images of major organs, including
the liver, spleen, kidneys, heart, lungs and brain collected from the
ꢀ1
control untreated mice and Et-IPCA-injected (25 mg kg ) mice at
various time points post-injection. No noticeable abnormality or lesion
was observed in any of the organs from the Et-IPCA-injected mice. All
scale bars are 100 mm.
Fig. 5 The blood biochemistry data of male ICR mice treated with CDs
at 100 mg kg and Et-IPCA at 25 mg kg on days 1, 7, 30 and 90. Age-
ꢀ1
ꢀ1
matched untreated mice were sacrificed on the same days as controls.
(
(
a–i) The time-course of the changes in the alanine aminotransferase
a), aspartate aminotransferase (b), alkaline phosphatase (c), total bili-
compared to those in the CDs group. Based on these ndings,
we concluded that the changes in Et-IPCA group might just be
a result of the continuous stimulation.
rubin (d), albumin (e), blood urea nitrogen (f), serum creatinine (g), total
cholesterol (h) and total triglycerides (i) in control mice, CDs-treated
mice and Et-IPCA treated mice. Each bar represents the mean ꢂ SD, n
¼
10. P* < 0.05 vs. control group. Gray bars indicate the range of values
3.5 Biodistribution of the CDs and Et-IPCA
obtained from healthy ICR mice.
To study the biodistribution characteristics of the CDs and Et-
IPCA in vivo, the emission spectra of tissue homogenates were
As was mentioned above, some factors examined in the obtained when the excitation wavelength was 360 nm. Then, the
biochemistry and hematology analyses changed signicantly in PL intensity at 450 nm was recorded because the curve reached
the CDs group, and most of which could be still be observed for its peak at this wavelength. As shown in Fig. 8, it was found that
up to 30 days aer injection. Among these changes, the blood both CDs and Et-IPCA mainly accumulated in reticuloendo-
cell counts, including the WBC, RBC and PLT, might indicate thelial system (RES) organs, especially in the liver, at three
that there was an inammatory response. Although the size of hours aer injection, which is similar to the in vivo behaviors of
our CDs is approximately 3 to 5 nm, the possibility of aggrega- many other small nanoparticles. There was also a considerable
tion in vivo should not be ignored. The aggregation of CDs may amount of nanoparticles found in serum and heart, indicating
form a carbon nanocluster whose size would be similar to that that both of the molecules could be easily transported by blood.
of viruses, which would affect the immune system. However, We can infer from the data obtained 24 h aer injection that the
these effects in the Et-IPCA group lasted a shorter time majority of both CDs and Et-IPCA had been excreted.
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Laboratory of Jilin Province and Preclinical Pharmacology R&D
Center of Jilin Province.
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9
In the present study, two strongly uorescent molecules, CDs
and Et-IPCA, with good photostability and biocompatibility
were successfully prepared. Systematic single- and repeated-
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