In vitro and in vivo toxicology of bare and PEGylated fluorescent carbonaceous nanodots in mice and zebrafish: the potential relationship with autophagy

Abstract

Fluorescent carbonaceous nanodots (CDs) are suitable for biomedical applications owing to their excellent photoluminescence properties. However, their toxicity has not been sufficiently evaluated. In this study, we demonstrated that both CDs and PEGylated CDs (PEG-CDs) displayed low cytotoxicity to several cell lines with a concentration as high as 1 mg mL⁻¹, and that autophagy might be involved in the cytotoxicity. In vivo, continuous administration of CDs or PEG-CDs showed no significant alteration on the hematology, blood biochemistry indexes and cell morphology of the main organs. In a zebrafish embryo model, CDs led to a considerably higher mortality and abnormality frequency than PEG-CDs, which demonstrated that PEGylation could decrease the toxicity of CDs. In conclusion, CDs displayed low systemic toxicity but considerable developmental toxicity, and PEGylation could reduce the toxicity, while autophagy may be involved in the toxic mechanisms of the CDs and PEG-CDs. Thus, our studies were exceedingly encouraging and provided some possibility for the clinical application of CDs, while developmental toxicity should be paid much more attention when evaluating the toxicity of nanomaterials.

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1. Introduction

Nanotechnology has rapidly developed in recent years, and it has provided various kinds of nanomaterials for disease diagnosis and drug delivery,^1,2 for example, nanomaterials for chemical drug delivery and gene delivery.^3–6 To fully address the requirements for disease diagnosis and drug delivery, nanomaterials are enabled with various distinct properties, including controllable size, high drug loading capacity, good targeting ability, good membrane penetration efficiency and a desirable drug release profile.^7–10 However, the toxicity of these nanomaterials is an increasing concern because safety is the first requirement if researchers want to clinically apply nanomaterials.^11–13 That is why only several nanomaterials have been approved by the food and drug administration of the government when so many kinds of nanomaterials have been developed. Knowledge about nanotoxicity could greatly improve the understanding of the nanomaterials currently in use.

Carbon-based nanomaterials have attracted great attention in the past decade because of their excellent properties for biotechnology, and various carbon-based nanomaterials were developed, including graphene, carbon nanotubes and carbonaceous nanodots (CDs), etc.^14–17 Among these, CDs were a new kind of nanomaterial that gained much attention due to their distinct advantages, including low cost and green synthetic route, seldom photobleaching, no optical blinking and low toxicity.^18,19 Nowadays, many methods such as thermal/hydrothermal oxidation, microwave/ultrasonic synthesis and electrochemical synthesis, have been developed to prepare CDs from various precursors such as silk, glucose, amino acids, juice and synthetic materials.^20–23 These CDs have been used for cell imaging and non-invasive in vivo imaging.^20–22 However, the toxicity of the CDs should be a primary concern if we want to apply them clinically. Although their cytotoxicity has been investigated in several studies, their long-term systemic toxicity in vivo, especially developmental toxicity, and potential mechanism are still unclear.

Autophagy plays a key role in helping cells survive diseases and stress conditions.²⁴ The primary function of autophagy is the degradation of cellular components (such as protein aggregates and defective cellular substructures) through lysosomal machinery.^24–26 However, autophagy blockade or autophagy induction may cause cytotoxicity due to the disturbance of a cell’s normal survival mechanism.^27,28 There have been reports of several nanomaterials that could induce or inhibit autophagy. For instance, graphene quantum dots could induce autophagy and lead to cell apoptosis.²⁹ Additionally, iron oxide nanoparticles,³⁰ cerium dioxide nanoparticles,³¹ gold nanoparticles,³² graphene oxide³³ and single-walled carbon nanotubes (SWCNTs)³⁴ could also induce autophagy which was related to the toxicity to cells. However, the potential role of CDs in inducing or inhibiting autophagy is still not clear.

In this study, we presented an overall evaluation of the in vitro and in vivo toxicity of CDs and PEG-CDs (Fig. 1). CDs were prepared by heat-treatment methods using glutamic acid as the only precursor.³⁵ Then the CDs were PEGylated (PEG-CDs) by procedures established by our group recently.³⁶ The cytotoxicity of CDs and PEG-CDs in many cell lines, and the relationship between cytotoxicity and autophagy were evaluated using MTT assay and a confocal laser scanning microscope. Long term toxicity to mice was evaluated by hematoxylin and eosin (H&E) staining, serum biochemistry tests and hematological analysis. Finally, zebrafish embryos were used to determine the developmental toxicity of CDs and PEG-CDs, which has been rarely evaluated and would provide more useful information to evaluate the possibility of the clinical application of carbonaceous nanodots.

Fig. 1 Overview of the in vitro and in vivo toxicology exploration of CDs and PEG-CDs. Cytotoxicity and cell autophagy are evaluated in vitro, while the long term toxicity, and developmental toxicity are assessed in vivo.

2. Results and discussion

2.1 Characterization of CDs

The hydrated particle sizes of CDs and PEG-CDs were 30.6 nm and 34.7 nm respectively, with zeta potentials of −6.7 mV and −10.6 mV respectively. However, according to the TEM images, the particle size was only approximately 3 nm (Fig. 2), which was consistent with our previous study.³⁵

Fig. 2 TEM images of CDs (a) and PEG-CDs (c), the scale bar represents 50 nm. The picture in the top right corner in each image represents the HRTEM image of the CDs (a) and PEG-CDs (c), the scale bar represents 2 nm. The particle size distribution (b and d) was determined using TEM.

The surface chemical groups of the CDs and PEG-CDs were characterized using high resolution X-ray photoelectron spectroscopy (XPS) (ESI, Fig. S1†). The C_1s spectrum of the CDs (ESI, Fig. S1a†) presented 4 peaks at 287.9, 286.6, 285.9 and 284.6 eV, which represent C=O, C–O, C–N and C–C respectively, while the C_1s spectrum of the PEG-CDs (ESI, Fig. S1d†) presented 3 peaks at 286.4, 285.4 and 284.6 eV, which represent C–O, C–N and C–C respectively.³⁷ The N_1s spectrum of the CDs (ESI, Fig. S1b†) presented 3 peaks at 401.4, 400.2 and 399.3 eV, which represent N–H, C–N and C–N–C respectively, while the N_1s spectrum of the PEG-CDs (ESI, Fig. S1e†) presented 2 peaks at, 400.1 and 398.7 eV, which represent C–N and C–N–C respectively.³⁸ The O_1s spectrum of the CDs (ESI, Fig. S1c†) presented 3 peaks at 535.4, 532.3 and 530.8 eV, which represent O=C–OH, C–OH/C–O–C and C=O respectively, while the O_1s spectrum of the PEG-CDs (ESI, Fig. S1f†) presented 2 peaks at 531.7 and 530.7 eV, which represent C–OH/C–O–C and C=O respectively.¹⁷ These results indicated that the surface of the CDs possessed abundant carboxyl and amidogen groups, while the PEGylation could cover these chemical groups.

2.2 Cytotoxicity

C6 cells were used for cell imaging in our previous study,³⁹ and in order to evaluate the cytotoxicity of CDs and PEG-CDs on more cell lines, two tumor cell lines (4T1 and C6) and two normal cell lines (human umbilical vein endothelial cell (HUVEC) and bEnd.3) were used, incubated with different concentrations of CDs and PEG-CDs for 24 h. Although a higher concentration of particles resulted in a lower cell viability (Fig. 3), the cell viability showed no significant difference between the control and cells treated with CDs and PEG-CDs at a concentration as high as 1 mg mL⁻¹, suggesting that the toxicity was low, which was consistent with previous studies.^35,36,40 However, elevating the concentration to 5 mg mL⁻¹ could lead to a significant decrease in the cell viability, indicating that a high concentration of CDs and PEG-CDs was harmful to cells. Anyway, the cytotoxicity of CDs and PEG-CDs was much lower than many other inorganic nanomaterials. For example, 150 nM of CdTe quantum dots significantly reduced the cell viability.⁴¹ Additionally, the cytotoxicity of CDs and PEG-CDs was also lower than other carbon-based nanomaterials. For instance, 1 mg mL⁻¹ of carboxylic acid functionalized SWCNTs significantly reduced the cell viability to approximately 50% ³⁴ and even 200 μg mL⁻¹ of graphene oxide could significantly inhibit cell proliferation.⁴²

Fig. 3 Cytotoxicity study of CDs and PEG-CDs on 4T1 (a), C6 (b), bEnd.3 (c) and HUVEC (d) cells (n = 6, mean ± SD).

2.3 Cell autophagy

Autophagy plays a vital role in cell survival, and both autophagy blockade and autophagy induction could cause cytotoxicity due to a disturbance of a cell’s normal survival mechanism.^27,28 To gain a better understanding of the relationship between autophagy and the toxicity of CDs and PEG-CDs, we investigated the autophagy induction effect using different concentrations of CDs and PEG-CDs. 4T1 cells (a murine mammary carcinoma cell line) were transfected with GFP-LC3 (GFP-labeled microtubule-associated protein 1A/1B-light chain 3) to construct an autophagy reporter cell line. LC3 is a protein-like ubiquitin in the autophagic stimulation; it was conjugated to phosphatidylethanolamine to form the LC3-II isoform, a marker for autophagy, on the surface of autophagosomes.⁴³ Thus, autophagy induction could be confirmed on the basis of the relative number of fluorescent autophagosomes (GFP-LC3 punctuations) formed. An appropriate concentration (50 μM) of hydroxychloroquine (HCQ) was used as the positive control because it can inhibit cell autophagy remarkably at the end of the autophagy process,⁴⁴ which would incite more fluorescent puncta than the control group (Fig. 4). Treating cells with CDs and PEG-CDs for 12 h caused obvious autophagosome accumulation (Fig. 4a), which was positively related to the concentration of CDs and PEG-CDs. The cells treated with CDs (1 mg mL⁻¹) exhibited a significant 8.0-fold increase in GFP-LC3 puncta compared with the control group, while the PEG-CDs (1 mg mL⁻¹) group showed a 5.9-fold increase in GFP-LC3 puncta compared with the control group. In addition, the CDs group showed a significant difference (p < 0.01) compared to the PEG-CDs group at a concentration of 1 mg mL⁻¹ (Fig. 4c). The HCQ (50 μM) plus CDs (0.04 mg mL⁻¹) group and HCQ (50 μM) plus PEG-CDs (0.04 mg mL⁻¹) group were used to find out the mechanism of the fluorescent autophagosome accumulation caused by CDs and PEG-CDs. Interestingly, the HCQ plus CDs group and HCQ plus PEG-CDs group presented a higher density of puncta than the HCQ group, CDs group and PEG-CDs group (Fig. 4a and c). Prolonging the incubation time to 24 h could further elevate the accumulation of autophagosomes and the quantitative result showed the same trend (Fig. 4b and d). These results demonstrated that both CDs and PEG-CDs could induce the formation of autophagosomes, which may be the reason for the toxicity of CDs and PEG-CDs and was consistent with many other studies.^32–34 The toxicity may be caused by the carboxyl groups beyond the CDs (ESI, Fig. S1†), while the PEGylation could cover the chemical group to reduce the toxicity. Other researchers also concluded that the toxicity of nanoparticles increases greatly when carboxyl groups are present on their surface.^45,46 Additionally, at the same concentration, PEG-CDs induced a lower autophagosome accumulation compared with CDs, which verified the lower toxicity of PEG-CDs compared with CDs. Similarly, Liu et al.³⁴ also reported that PEGylation could reduce the accumulation of autophagosomes caused by carboxylic acid functionalized single-walled carbon nanotubes. Some other research also showed that cationic polyamidoamine dendrimers are more prone to inducing autophagy in cells than anionic PAMA,⁴⁷ demonstrating that surface groups may be an important factor in inducing autophagy. In our previous study, we found that the surface of bare CDs was rich with NH₂ and COOH,³⁵ while PEGylation could cover these groups and bring about a neutral surface. This may explain the lower toxicity of PEG-CDs.

Fig. 4 Autophagy inhibition by different concentrations of CDs and PEG-CDs in GFP-LC3 transfected 4T1 cells after 12 h (a) and 24 h (b) incubation. Scale bar represents 10 μm. Graphs shows mean ± SD puncta per cell after 12 h (c) and 24 h (d) incubation. Data presented is mean ± SD (n = 20). *, ** and *** in the column diagram represent p < 0.05, p < 0.0 and p < 0.001 respectively versus the control group, and the *, ** and *** in the transverse line represent p < 0.05, p < 0.01 and p < 0.001 respectively between the compared groups; n.s means no significant difference.

2.4 In vivo toxicity

To further evaluate the toxicity of CDs and PEG-CDs, a multi-dose was applied because a single dose could not reflect the potential clinical dose of these particles.⁴⁸ Mice received 5 mg kg⁻¹ of CDs or PEG-CDs everyday through the tail vein. During the treatment, no obvious changes in body weights were observed, suggesting that both CDs and PEG-CDs had no significant influence on the mice body weights (Fig. 5). Hematology and blood biochemistry analysis were carried out at 7 days and 21 days after the first dose, respectively. Most of the hematology indexes were within the normal range (Fig. 6), including the white blood cell (WBC), red blood cell (RBC), hematocrit (HCT), mean corpuscular volume (MCV), hemoglobin (HGB), platelet (PLT), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values. The WBC count is sensitive to the physiological conditions. Although the WBC count of the PEG-CDs was higher than that of the control on the 7th day (Fig. 6a), it returned to a normal range on the 21st day (Fig. 6b). On the 21st day, the WBC count of the CDs group was higher than that of the control (Fig. 6b), but it was still within the normal level. However, a similar cumulative dose of graphene oxide could cause a significant decrease in the WBC count 1 month after administration.⁴⁸ A single high dose (100 mg kg⁻¹) of graphene oxide could also result in a decrease in the WBC count, although it could be recovered on the 21st day after administration.⁴⁹ These results demonstrated that CDs and PEG-CDs possessed low hematology toxicity.⁴⁰

Fig. 5 Body weight of mice during treatment (n = 6, mean ± SD).

Fig. 6 Hematology data of mice treated with CDs (5 mg kg⁻¹), PEG-CDs (5 mg kg⁻¹) and saline at 7 days (a) and 21 days (b) after the first dose (n = 6, mean ± SD).

Additionally, several blood biochemistry indexes were determined to reflect the toxicity to several tissues, such as total protein (TP), albumin (ALB), globulin (GLOB), albumin/globulin (A/G), alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and creatinine (CREA). TP, ALB, GLOB, ALT and AST could reflect the toxicity to the liver. All of these five parameters were within the normal range (Fig. 7), suggesting that the repeated injection of CDs and PEG-CDs displayed no harmful effect on the liver. BUN and CREA, two indexes of the toxicity to the kidney, were also within the normal range after treatment with CDs and PEG-CDs (Fig. 7), suggesting that CDs and PEG-CDs possessed no significant kidney toxicity. Other carbon-based nanomaterials also showed low in vivo toxicity. For example, a single dose of graphene (20 mg kg⁻¹) or graphene oxide (100 mg kg⁻¹) did not cause significant toxicity to the liver and kidney,⁵⁰ and repeated administration of graphene quantum dots did not cause obvious modification on blood biochemistry indexes either.¹³

Fig. 7 Blood biochemistry indexes of mice treated with CDs (5 mg kg⁻¹), PEG-CDs (5 mg kg⁻¹) and saline at 7 days (a) and 21 days (b) after the first dose (n = 6, mean ± SD).

Haematoxylin and eosin staining (H&E staining) of the heart, kidney and liver was used to further determine the toxicity of CDs and PEG-CDs (Fig. 8). Cells in all of the slices exhibited normal morphology, suggesting that CDs and PEG-CD would not induce organic harm to these three tissues. Combining this with the hematology and blood biochemistry analysis, we could conclude that both CDs and PEG-CDs showed low long-term toxicity to mice, which was useful for clinical applications in the future. A study performed by Chong et al. showed that the long-term toxicity of graphene quantum dots was lower than that of graphene oxide,⁴⁸ suggesting that carbon-based dots were biocompatible.

Fig. 8 H&E staining of heart, kidney and liver from mice treated with CDs (5 mg kg⁻¹), PEG-CDs (5 mg kg⁻¹) and saline at 7 days (a) and 21 days (b) after the first dose. Scale bar represents 50 μm.

2.5 Developmental toxicity

The developmental toxicity of CDs and PEG-CDs was evaluated by treating zebrafish embryos at 6 hours post-fertilization (hpf) with different concentrations (10, 50 and 100 μg mL⁻¹) of CDs and PEG-CDs. The mortality rate was determined at 24 hpf (Fig. 9a). The zebrafish embryos treated with low concentrations (10 and 50 μg mL⁻¹) of CDs and PEG-CDs showed no significant difference compared with the control group. However, 100 μg mL⁻¹ of CDs led to 100% mortality while for 100 μg mL⁻¹ of PEG-CDs, it was only 32.5%, suggesting that the higher concentration of CDs could result in significant developmental toxicity and PEGylation could reduce the toxicity. However, the toxicity of other inorganic nanomaterials was much higher. For example, incubation with silver nanoparticles at a concentration as low as 8 pM for only 2 h could result in 63% mortality of zebrafish embryos.⁵¹ Additionally, 20 μM of PEGylated CdSe/ZnS quantum dots caused only 10% of embryos to die, but the same concentration of oxidative weathered quantum dots caused about 60% death,⁵² suggesting that PEGylation could reduce the toxicity of particles, which was consistent with our study. The phenotype of the zebrafish embryos was observed 48 hpf with an emphasis on the extent of abnormalities. We only measured the abnormality rate of the control group and groups treated with 10 and 50 μg mL⁻¹ of CDs and PEG-CDs because the embryos treated with 100 μg mL⁻¹ of CDs all died at 24 hpf (Fig. 9a). The result suggested that the developmental abnormalities of CD-treated embryos were much higher than those treated with PEG-CDs (Fig. 9b). At 50 μg mL⁻¹, the abnormality frequency of CD-treated embryos was 2.3-fold higher than those treated with PEG-CDs. Representative pictures could also show the abnormality of CD- and PEG-CD-treated embryos (Fig. 9c). These results strongly demonstrated that the CDs displayed developmental toxicity, which was positively related to concentration, and it was consistent with other nanomaterials.^52,53 However, PEGylation could obviously decrease the developmental toxicity.⁵² Comparatively, even 0.01 mM of gold nanoparticles and 500 pM of quantum dots could induce the phenotype abnormality of zebrafish embryos.^54,55 Carbon-based nanomaterials showed a lower toxicity. As high as 40 μg mL⁻¹ of multi-walled carbon nanotubes (MWCNTs) showed no obvious effect on the phenotype of zebrafish. However, 60 μg mL⁻¹ of MWCNTs could cause significant phenotypic defects.⁵⁶ In our study, 50 μg mL⁻¹ of CDs caused only 9% of zebrafish abnormality, suggesting that CDs possessed a lower toxicity on the development of zebrafish embryos compared with other carbon-based nanomaterials.

Fig. 9 Mortality rate (a), abnormality rate (b) and phenotype (c) of the zebrafish embryos treated with CDs and PEG-CDs. CDs and PEG-CDs were added to zebrafish embryos at 6 hpf. The mortality rate was measured at 24 hpf, the abnormality rate was measured at 48 hpf and the phenotype was observed at the same time point. Scale bar represents 100 μm. Data show mean ± SD. Three independent experiments were carried out.

3. Experimental

3.1 Materials

Glutamic acid (purity > 98.5%) was purchased from Sinopharm Chemical Reagent (Shanghai, China). 4T1, C6, bEnd.3 and HUVEC were purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Hydroxychloroquine sulfate (HCQ (purity > 98.0%)) was purchased from TCI Development Co., Ltd. (Shanghai, China). Amino poly-(ethylene glycol) (NH₂–PEG, MW = 5000, purity > 95.0%) was purchased from Seebio Biotech, Inc (Shanghai, China). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Plastic cell culture dishes and plates were obtained from Wuxi NEST Biotechnology Co. Ltd. (Wuxi, China). Dulbecco’s Modified Eagle Medium (high glucose) cell culture medium (DMEM) and FBS were obtained from Life Technologies (Grand Island, NY, USA). eGFP-LC3 plasmid was obtained from Addgene. Other chemicals and reagents were of analytical grade. Kunming mice (male, 4–5 weeks, 18–22 g) were obtained from Dashuo Biotechnology Co., Ltd (Chengdu, China) and maintained under standard housing conditions. Zebrafish embryos of wild-type AB and cardiac myosin light chain 2 (cmlc2) transgenic zebrafish (Tg (cmlc2:EGFP)) were kindly offered by Doctor Huaqin Sun (Joint Laboratory of Reproductive Medicine, Sichuan University, The Chinese University of Hong Kong).

3.2 Synthesis and characterization

The CDs were synthesised according to our previous study.³⁵ 1 g of glutamic acid was added into the flask which was heated to 280 °C, in order to carbonize the glutamic acid, the heater was kept for 1 min. After the flask cooled to 70 °C, 5 mL of deionized water was added. The solution was sonicated for 10 min, followed by 30 min of centrifugation at 10 000 rpm, then the CDs in the supernatant were harvested. In order to realise PEG conjugation, the carboxyl groups of the CD solution (20 mg mL⁻¹) were activated by NHS and EDC in PBS for 0.5 h. Then, NH₂–PEG (80 μg mL⁻¹) was added to the solution. After stirring for 2 h, the unconjugated PEG was removed by dialysis (cut-off size = 10 kDa). The hydrated diameter and zeta potential were measured using a Malvern Zetasizer (Malvern, NanoZS, UK). Transmission electronic microscopy (TEM) images were obtained using a JEOL JEM-2010 microscope (Japan) operated at 200 kV. XPS experiments were carried out using an AXIS Ultra DLD (Kratos UK) according to the standard protocols of our previous study,²⁰ and the data were analyzed using XPS PEAK 4.1 software.

3.3 Cytotoxicity

The cytotoxicity of the CDs and PEG-CDs was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 4T1 (mice mastadenoma), C6 (Murine glioma), HUVEC (Human Umbilical Vein Endothelial) and bEnd.3 (murine brain endothelial) cells were seeded in 96-well plates at a density of 2 × 10³ cells per well. C6 cells and 4T1 cells were cultured in RPMI-1640, HUVEC and bEnd.3 cells were cultured in DMEM, and the culture media were supplemented with 10% FBS, 100 U mL⁻¹ streptomycin, and 100 U mL⁻¹ penicillin, in an atmosphere of 5% CO₂ at 37 °C. Twenty four hours later, the CDs and PEG-CDs were diluted in the culture media without FBS and added into the wells in a series of concentrations ranging from 0.0016 mg mL⁻¹ to 5 mg mL⁻¹. After incubation for 24 h, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg mL⁻¹) was added into each well and incubated for another 4 h. Then the medium was replaced by dimethyl sulfoxide and the absorption at 490 nm was observed using a microplate reader (Thermo Scientific Varioskan Flash, USA).

3.4 Cell autophagy

The GFP-LC3 4T1 cell line was constructed using the GFP-LC3 plasmid vector. The vector contained a cDNA fragment related to an autophagy marker called LC3, which expressed an GFP-LC3 fusion protein. eGFP-LC3 expressing 4T1 cells were harvested by eGFP-LC3 plasmid transfection. In brief, cells were seeded in a coverslip in 6-well plates at a density of 1 × 10⁴ per single well with 2 mL of RPMI-1640 (without antibiotics). Then we transferred the plasmid into the 4T1 cell line using Lipofectamine 2000 according to the manufacturer’s instructions. Pooled GFP-LC3 plasmid (14 μg) was diluted in OptiMEM (700 μL) for each well (6-wells). 5 min later, the diluted DNA and Lipofectamine 2000 were mixed and incubated for 10 min (room temperature). Then 250 μL of the plasmid mixture was added to the well. After 24 h incubation, the GFP-LC3-expressing cells were harvested. Then the cells were treated with different concentrations of CDs and PEG-CDs, and HCQ (50 μM) was used as the positive control. 12 h or 24 h later, the cells were washed and fixed with 4% (vol/vol) paraformaldehyde. Images were captured using a confocal laser scanning microscope (LSM710, Carl Zeiss, Germany). To get the statistical data of the autophagosome accumulation in the cells, the number of bright green punctuations (autophagosomes) was calculated in at least 20 cells.

3.5 In vivo toxicity

Long-term in vivo toxicity was determined by the body weight variation, hematology and blood biochemistry indexes and H&E staining. All animal experiments were carried out in accordance with protocols evaluated and approved by the ethics committee of Sichuan University. Thirty-six mice were randomly divided into 3 groups: saline group, CDs (5 mg kg⁻¹) group and PEG-CDs (5 mg kg⁻¹) group. The body weights were recorded every 2 days. 7 days and 21 days after the administration, whole blood and serum were collected from 6 mice from each group for hematology and blood biochemistry analysis using an MEK-6318K automated hematology analyzer (Nihon-kohden, Shinjuku-ku, Tokyo, Japan) before being sacrificed after heart perfusion by PBS. Major organs (liver, kidney and heart) from those mice were harvested and applied for H&E staining according to standard protocols provided by the manufacturers. 10 μm sections from at least three different planes of the organs were cut and used for H&E staining. Sections were evaluated using and examined by a digital microscope at various magnifications within different fields.

3.6 Developmental toxicity

Zebrafish embryos of wild-type AB were gathered from the zebrafish aquarium in the Joint Laboratory of Reproductive Medicine, Sichuan University, The Chinese University of Hong Kong and were staged according to standard procedures. In brief, zebrafish strains were raised in an incubator at 28 °C in a 14 h light and 10 h dark cycle using UV treated and aerated water.⁵⁷ Fifteen of the 6 hour post-fertilization (hpf) zebrafish embryos were added into each well of 24-well plate, followed by the CDs and PEG-CDs with different concentrations (10 μg mL⁻¹, 50 μg mL⁻¹, 100 μg mL⁻¹). The mortality of each well was determined at 24 hpf using a stereoscopic microscope (SMZ1000, Nikon, Japan), and phenotype abnormalities were observed using a stereoscopic microscope (SMZ1000, Nikon, Japan) at different time points (24 and 48 hpf). Three independent experiments were carried out.

3.7 Statistical analysis

All of the values were presented as mean ± SD. A statistical comparisons method used was two-tailed student’s t test for all of the data and a p value of < 0.05 was considered an indication of statistical significance. The number of GFP-LC3 bright green punctuations was analyzed using ImageJ Software.

4. Conclusion

In our study, CDs and PEG-CDs at a dose of 5 mg kg⁻¹ showed low systemic toxicity in vivo through blood biochemistry, hematology, and histology analysis. Although CDs possessed slight developmental toxicity, PEGylation could further reduce the toxicity. Additionally, both CDs and PEG-CDs could induce the autophagy of cells, which may be the reason for the toxicity. Although, more toxicology studies of CDs at higher doses and the use of more animal models are necessary before any potential clinical applications of this material. However, our studies are extraordinarily encouraging and provide some possibility for future carbonaceous nanodot-based clinical research.

Acknowledgements

The work was granted by the National Natural Science Foundation of China (81402866, 81301974) and the Sichuan University Starting Foundation for Young Teachers (2014SCU11044).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra05201g

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