Cytotoxicity and transcriptome changes triggered by CuInS2/ZnS quantum dots in human glial cells.

As a newly developed cadmium-free quantum dot (QD), CuInS2/ZnS has great application potential in many fields, but its biological safety has not been fully understood. In this study, the in vitro toxicity of CuInS2/ZnS QDs on U87 human glioma cell line was explored. The cells were treated with different concentrations of QDs (12.5, 25, 50 and 100 µg/mL), and the uptake of QDs by the U87 cells was detected by fluorescence imaging and flow cytometry. The cell viability was observed by MTT assay, and the gene expression profile was analyzed by transcriptome sequencing. These results showed that QDs could enter the cells and mainly located in the cytoplasm. The uptake rate was over 90 % when the concentration of QDs reached 25 µg/mL. The cell viability (50 and 100 µg/mL) increased at 24 h (P < 0.05), but no significant difference after 48 h and 72 h treatment. The results of differential transcription showed that coding RNA accounted for the largest proportion (62.15 %), followed by long non-coding RNA (18.65 %). Total 220 genes were up-regulated and 1515 genes were down-regulated, and significantly altered gene functions included nucleosome, chromosome-DNA binding, and chromosome assembly. In conclusion, CuInS2/ZnS QDs could enter U87 cells, did not reduce the cell viability, but would obviously alter the gene expression profile. These findings provide valuable information for a proper understanding of the toxicity risk of CuInS2/ZnS QD and promote the rational utilization of QDs in the future.

PEGylated CuInS2/ZnS quantum dots inhibit neurite outgrowth by downregulating the NGF/p75NTR/MAPK pathway.

The widespread application of cadmium-free CuInS2/ZnS QDs has raised great concern regarding their potential toxicity to humans. To date, toxicological data related to CuInS2/ZnS QDs are scarce. Neurons play extraordinary roles in regulating the activities of organs and systems, and serious consequences occur when neurons are damaged. Currently, the potential toxicity of CuInS2/ZnS QDs on neurons has not been fully elucidated. Here, we investigate the neurotoxicity of PEGylated CuInS2/ZnS (CuInS2/ZnS-PEG) QDs on neuron-like PC12 cells. We found that CuInS2/ZnS-PEG QDs were taken up by PC12 cells, but at a concentration range from 0 to 100 µg/mL, they did not affect the survival rate of the PC12 cells. In addition, we found that CuInS2/ZnS-PEG QDs significantly inhibited neurite outgrowth from and the differentiation of PC12 cells in the presence of NGF, while COOH-modified CuInS2/ZnS QDs or free PEG did not have a similar effect. Further studies showed that CuInS2/ZnS-PEG QDs obviously downregulated the expression of low-affinity NGF receptor (p75NTR) and subsequently negatively regulated the downstream MAPK cascade by dephosphorylating ERK1/2 and AKT. Taken together, these results suggest that CuInS2/ZnS-PEG QDs disturb NGF signal transduction from external stimuli to relevant internal signals, thus affecting normal biological processes such as neurite outgrowth and cell differentiation.

Cytotoxicity and Immune Dysfunction of Dendritic Cells Caused by Graphene Oxide.

Graphene, known as "black gold", has important applications in various fields. In previous studies, it has been proved that graphene oxide (GO) which is a derivative of graphene has low toxicity. However, the immunotoxicity of GO has not been fully elucidated. In this work, we used DC2.4 cell line to investigate the in vitro immunotoxicity of two types of GO, mono-layer GO (mono-GO) and multi-layer GO (multi- GO). We found that mono-GO had less effect on cell viability than multi-GO, but both mono-GO and multi-GO significantly induced the generation of ROS in DC2.4 cells. Interestingly, mono-GO caused DC2.4 cells to aggregate, thus changed the cell morphology significantly. However, no similar influence occurred for multi-GO. In addition, the results showed that these two GOs obviously enhance the release of TNF-α by DC2.4 cells with and without LPS stimulation. GO did not affect the level of IL-6 released from DC2.4 cells, but multi-GO promoted the release of IL-6 while mono-GO inhibited the production of IL-6 when cells were in response to LPS stimulation. Whole-transcriptome sequencing analysis found some immune-related differentially expressed genes including H2-DMb1, Ncbp3, Oas2, Men1, Fas, Cd320, Cd244, and Tinagl1 which are engaged in the immune system process. These results suggested that both mono-GO and multi-GO are immunotoxic to DC2.4 cells, which provides important basis for subsequent biological and clinical medical applications.

Synthetic and immunological studies on the OCT4 immunodominant motif antigen-based anti-cancer vaccine.

Objective: Cancer stem cell is one of the important causes of tumorigenesis as well as a drug target in the treatment of malignant tumor. However, at present, there is no immune vaccine targeting these cells. Octamer-binding transcription factor 4 (OCT4), a marker of embryonic stem cells and germ cells, often highly expresses in the early stages of tumorigenesis and is therefore a good candidate for cancer vaccine development. Methods: To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked three different OCT4 epitope antigens to a carrier protein, keyhole limpet hemocyanin (KLH), combined with Toll-like receptor 9 agonist (TLR9). Results: Immunization with OCT4-3 + TLR9 produced the strongest immune response in mice. In prevention assays, significant tumor growth inhibition was achieved in BABL/c mice treated with OCT4-3 + TLR9 (P < 0.01). Importantly, the results showed that cytotoxic T lymphocyte activity and the inhibition of tumor growth were enhanced in mice immunized with OCT4-3 combined with TLR9. Meanwhile, multiple cytokines [such as interferon (IFN)-γ (P < 0.05), interleukin (IL)-12 (P < 0.05), IL-2 (P < 0.01), and IL-6 (P < 0.05)] promoting cellular immune responses were shown to be greatly enhanced in mice immunized with OCT4-3 + TLR9. Moreover, we considered safety considerations in terms of the composition of the vaccines to help facilitate the development of effective next-generation vaccines. Conclusions: Collectively, these experiments demonstrated that combination therapy with TLR9 agonist induced a tumor-specific adaptive immune response, leading to the suppression of primary tumor growth in testis embryonic carcinoma.

In vitro and in vivo immunotoxicity of PEGylated Cd-free CuInS2/ZnS quantum dots.

The annual increase in the production and the use of engineering quantum dots (QDs) have led to concern about exposure and safety of QDs. To resolve the risk of Cd release from QDs, a series of Cd-free QDs, represented by CuInS2/ZnS QDs, has been developed in recent years. However, the toxicological profile of CuInS2/ZnS QDs has not been fully elucidated, especially, their immunotoxicity. Here, we performed a detailed in vitro cytotoxicity study on PEGylated CuInS2/ZnS QDs using the DC2.4 cell line and investigated their in vivo immunotoxicity using BALB/c mice. In vitro experiments showed that CuInS2/ZnS QDs were taken up by cells, promoted cell viability, enhanced release of tumor necrosis factor-α, and decreased the level of interleukin (IL)-6 in response to lipopolysaccharide stimulation. More than 5000 genes at the transcriptome level were observed by high-throughput RNA sequencing after CuInS2/ZnS QD exposure. In vivo study showed that CuInS2/ZnS QDs increased the levels of IL-4 on day 1 and enhanced the levels of IL-10 and IL-13 on day 28 in mice. There was no obvious difference in the number of spleen-derived lymphocytes, organic index, hematology and immune organ histology on days 1 and 28 after treatment. These findings demonstrated that PEGylated CuInS2/ZnS QDs disturbed the function of DC2.4 immune cells in vitro, but caused no obvious toxicity to immune system in vivo, suggesting that PEGylated CuInS2/ZnS QDs are biocompatible and have potential for bioapplication in the future.

In Vivo Toxicity Evaluation of PEGylated CuInS2/ZnS Quantum Dots in BALB/c Mice.

In recent years, quantum dots (QDs) have emerged as a potential contrast agent for bioimaging due to their bright luminescence and excellent photostability. However, the wide use of QDs in vivo has been limited due to underlying toxicity caused by leakage of heavy metals. Although non-cadmium QDs have been developed to resolve this issue, a comprehensive understanding of the toxicity of these newly developed QDs remains elusive. In this study, we administered PEGylated copper indium sulfide/zinc sulfide (CuInS2/ZnS), which are typical non-cadmium QDs, and analyzed the long-term effects of these nanoparticles in BALB/c mice. Body weight, hematology, blood biochemistry, organ histology, and biodistribution were examined at different time points. We found no significant difference in body weight after injection of CuInS2/ZnS QDs. These CuInS2/ZnS QDs entered and were accumulated in major organs for 90 days post-injection. The majority of biochemical indicators were not significantly different between the QDs-treated group and the control group. In addition, no significant histopathological abnormalities were observed in the treated mice compared with the control mice. CuInS2/ZnS QDs did not lead to observable toxicity in vivo following either the administration of a high or low dose. Our research not only provides direct evidence of the bio-safety of CuInS2/ZnS QDs, but also a feasible method for evaluating nanoparticle toxicity.

Synergistic antitumor activity of TRAIL combined with chemotherapeutic agents in A549 cell lines in vitro and in vivo.

PURPOSE: To investigate the synergistic cytotoxicity of TRAIL in combination with chemotherapeutic agents in A549 cell lines, we systematically evaluated the cytotoxicity of TRAIL alone and TRAIL in combination with cisplatin, paclitaxel (Taxol) or actinomycin D in A549 cell lines in vitro and in vivo, and whether the sensitivity was correlated with the expression level of TRAIL receptors. METHODS: We investigated the cytotoxicity of TRAIL alone and the synergistic antitumor effects of TRAIL in combination with chemotherapeutic agents in A549 cells by crystal violet staining and FACS in vitro. The expression levels of DR4, DR5, DcR1 and DcR2 were measured in TRAIL-treated and chemotherapeutic agent-treated A549 cells by Western blotting. The growth inhibition of tumors was evaluated in terms of incidence, volume and weight in a A549-implanted nude mice model. RESULTS: Chemotherapeutic agents cisplatin (5.56 mug/ml), Taxol (10 and 30 mug/ml) or actinomycin D (9.26, 83.3 and 750 ng/ml) augmented the cytotoxicity of TRAIL in A549 cell lines within a range of concentrations of TRAIL (1.98-160 ng/ml) in vitro. The expression levels of DR4 and DR5 were not significantly different and the expression of DcR2 was slightly downregulated, but the expression of DcR1 was not detected in non-treated, TRAIL-treated and chemotherapeutic agent-treated A549 cells. The rates of tumor inhibition following treatment with TRAIL alone (15 mg/kg per day, daily for 10 days) and TRAIL/cisplatin (15 mg/kg per day TRAIL, daily for 10 days; 1.5 mg/kg per day cisplatin, daily for 10 days with 7-day intervals) were 28.3% and 76.8% by tumor weight ( P<0.05 for TRAIL alone versus control, P<0.05 for TRAIL/cisplatin versus cisplatin alone and TRAIL alone) on day 65 in vivo. CONCLUSION: TRAIL in combination with chemotherapeutic agents cisplatin, Taxol or actinomycin D exerted synergistic antitumor effects in A549 cell lines in vitro and TRAIL/cisplatin demonstrated synergistic antitumor effects in vivo. The expression levels of TRAIL receptors suggested that the synergistic effects of TRAIL in combination with chemotherapeutic agents are not at the receptor level in A549 cell lines.