Therapeutic effect of quantum dots for cancer treatment

Abstract

Semiconductor quantum dots (Qdots) are well established as a unique category of fluorescent imaging probes due to their superior optical properties over conventional small-molecule dyes. At the same time, there have been major concerns regarding their potential nano-toxicity because high-quality Qdots often contain heavy metal elements. Here, we explore the possibility of converting this drawback for therapeutic applications. Using a human liver hepatocellular carcinoma model, human hepatocyte line model, and the Henrietta Lacks strain of cancer cells, we show that tumour cell growth is inhibited with an IC₅₀ value in the μM range under in vitro conditions. Furthermore, under in vivo conditions, the mean survival time of tumour-bearing mice can be extended by 2.5 times when treated with Qdots. These results demonstrate the possibility of converting nano-toxicity to antitumour activity.

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Quantum dots (Qdots) have become an indispensable tool in fluorescence bioimaging, especially for applications requiring high sensitivity, high multiplexing, and extended imaging time.^1–5 In the meantime, their potential toxicity has also been the centre of debate because of the heavy metal compounds often included in high-quality particles. In many cell lines, Qdots' cytotoxicity have been observed through various molecular mechanisms including generation of reactive oxygen species (ROS), mitochondrial and DNA damage;^6–8 and the toxicity has been correlated with particle size, shape, surface charge, colloidal stability, and coating material.^9–12 Under in vivo conditions, signs of toxicity are generally not obvious due to the relatively short experiment periods (months).^13,14 This is perhaps not very surprising because advanced surface coating materials such as amphiphilic polymers (non-biodegradable) are commonly used to protect the integrity of Qdots.^15–17 Nevertheless, the long-term toxicity (years or decades) of Qdots after the coating materials and core particle breakdown remains a major concern.

Here, we investigate the possibility of converting this major toxicity problem to a new class of cancer therapeutic. To make water soluble Qdots and promote their endocytosis, we coated a layer of polyamines (PA) (see Scheme S1 in the ESI†) to Qdot surface (Fig. 1a). It is widely known that cationic nanoparticles have high affinity to negatively charged cell surface and high endocytosis rate.^18–21 Furthermore, tumour cells are generally hungrier for polyamines than normal cells due to upregulated polyamine transporter (PAT).^22–24 Despite this enhanced uptake in tumour cells, it is worth mentioning that more selective tumour cell targeting can be achieved by linking Qdots with a specific targeting ligand (e.g., antibody and aptamer), but it is not the focus of this work, which is aimed at evaluating Qdot toxicity for tumour treatment.

Fig. 1 Qdots coated with polyamines (PA) and their structural and spectroscopic characterizations. (a) Schematic illustration PA-coated Qdots; (b) TEM showing CdSe/ZnS@PA (4.8 ± 0.2 nm); (c) UV-vis and luminescence spectra of Qdots.

We first characterized the structural and optical properties of the as synthesized Qdots. Transmission electron microscopy (TEM) micrograph shows that the Qdots maintained the same size, shape, and dispersity after ligand exchange (Fig. 1b); whereas UV-vis and fluorescence spectroscopy measurements show sharp absorption and emission peaks centred at 595 and 613 nm, respectively (Fig. 1c). The PA-coated CdSe/ZnS Qdots (CdSe/ZnS@PA) are highly emissive with a quantum yield (Q.Y.) of 67.4 ± 0.6%, which were measured relative to the value of rhodamine B (Q.Y. = 89% in EtOH at room temperature) as the reference.

Next, we characterized Qdots' cellular uptake in a pair of human liver cells (HepG2, a well-differentiated hepatocellular carcinoma, and QSG-7701, a human hepatocyte line) using fluorescence microscopy, flow cytometry, and TEM. Qualitative confocal imaging shows substantially more Qdot uptake in HepG2 cells compared to QSG-7701 under the same treatment condition (Fig. 2a), likely due to the upregulated PAT in tumour cells as aforementioned. Quantitative flow cytometry experiment confirms the enhanced uptake in HepG2 and shows a 10-fold increase (Fig. 2b). Intracellular distribution of the Qdots can be visualized using TEM. As shown in Fig. 2c, Qdots are largely clustered in endosome within the first 3 h of incubation, and begin to escape into the cytosol after ∼6 h due to endosomal membrane damage. This is likely a result of the strong electrostatic interaction between the cationic Qdot surface and the anionic vesicle membrane,^25,26 as well as the proton buffering capability of amines.^27,28

Fig. 2 Cellular uptake of Qdots. (a) Confocal images, (b) flow cytometry diagram; and (c) TEM images of living cells (HepG2) loaded with 2.5 μM Qdots (3 h and 6 h incubation). (d) IC₅₀ value comparison of Qdots in HepG2 and QSG-7701 cells. QSG-7701 cells were used as the control.

To investigate the cell growth inhibition capability of Qdots, HepG2, QSG-7701 and HeLa cells were incubated with various concentrations of Qdots for 48 h. The IC₅₀ values of Qdots measured by the MTT assay at 48 h are ∼2.51 μM in HepG2 cells, ∼26.65 μM in QSG-7701 cells, and ∼2.67 μM in HeLa cells, respectively (Fig. 2d and Table S1†). The approximately 11-fold difference reflects the differential Qdot uptake capabilities between the two cell lines, indicating preferential cell killing in cancer cells, a desired property that can be further improved by using more selective targeting ligands (e.g., antibodies).

To understand the underlying molecular mechanisms of the antitumour effect, we proceeded with a series of imaging assays to examine key biomarkers in cells and detailed cell structures. Several lines of evidence suggest that the antitumour effect arises from ROS-induced cell apoptosis. We first investigated whether Qdots could increase the ROS level in HepG2 cells by confocal microscopy and flow cytometry. In our experiment, ROS accumulation was quantified by the 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) assay. Confocal microscopic analysis of DCF-stained Qdots-treated cells shows a significant increase in intensity of DCF staining compared with the control cells (Fig. 3a, right). That is, the intracellular ROS levels were significantly increased in HepG2 cells treated with Qdots. The results were further confirmed by flow cytometry results (see Fig. S2 in the ESI†), indicating that the mitochondrial dysfunction was likely related to the production of ROS.

Fig. 3 Cell apoptosis induced by Qdots. (a) Confocal microscopy results of ROS productions after HepG2 cells treated with Qdots for 24 h (excitation: 488 nm, and emission: 500–560 nm). (b) Cell morphological changes caused by Qdots indicated by Hoechst staining (excitation: 405 nm, emission: 430–480 nm). (c) Confocal images, and (d) flow cytometry data of HepG2 cells treated with Qdots. Cells are stained with annexin V-FITC. (e) Fluorescence imaging of JC-1 labeled HepG2 cells for analysis of MMP by confocal microscopy. (f) Representative TEM images showing the ultra-structure of HepG2 cells after treatment with Qdots (2.5 μM) for 48 h. Normal HepG2 cells are typically round with their surface covered with microvilli projecting in all directions; whereas in apoptotic cells the nuclei (N) are irregularly shaped, the mitochondria (#) are swollen and unstructured, and the chromatins (*) are condensed. (g) Caspase 3 activity after Qdots treatment for 12 h at indicated concentrations.

Cell morphological change incurred during apoptosis is another signature of cell death.^29–31 To observe the morphologic characteristics of apoptotic nuclei, HepG2 cells were stained with Hoechst 33342 after exposure to Qdots for 12–48 h and imaged by fluorescence microscopy. Control cells exhibited homogeneous nuclear staining, but apoptotic cells displayed reduction of cellular volume, fragmented nuclei, and condensed chromatin, typical characteristics of apoptosis (Fig. 3b). The number of dead cells also increased gradually in a time-dependent manner with extended incubation with Qdots. In comparison, QSG-7701 cells treated with Qdots show no obvious signs of apoptosis, due to lower level of Qdots uptake.

To distinguish cells in early stages of apoptosis from necrotic cells, cells were stained with annexin V.^32,33 Annexin V binds to the membrane phospholipids phosphatidylserine (PS), which is externalized from the inner to the outer surface of the plasma membrane in early-stage apoptosis.^34–36 Using annexin V-FITC, the staining of HepG2 cells treated with Qdots show intense fluorescence compared to control cells (Fig. 3c), confirming early-stage apoptosis. Flow cytometric analysis based on Annexin V-positive staining revealed that the percentage of apoptotic cells in Qdots-treated HepG2 cells was ∼70.6% (Fig. 3d). In contrast, the percentage of apoptosis in QSG-7701 cells was approximately 10-fold less (∼7.1%, Fig. 3d).

The activation of cysteine proteases (caspases) is another established biochemical character for both early and late stages of apoptosis.³⁷ HepG2 cells were treated with Qdots at a series of concentrations for 12 h, after which the caspase 3 activity was evaluated by using the Caspase-Glo assay. With increasing the concentration of Qdots, the caspase 3 activity in HepG2 cells rises accordingly (Fig. 3g). For a further assessment of apoptosis induced by Qdots, we examined the sub-cellular structure using TEM. Fig. 3f shows the representative TEM images of the control cells and the cells treated with Qdots. Large numbers of microvilli were observed in the cytoplasm of the control cells, whereas abnormal nuclear shapes, clumping chromatin condensations, and swollen and unstructured mitochondria were noticed in the Qdots-treated HepG2 cells (Fig. 3f). These observations support the hypothesis that Qdots can induce apoptosis in cells above a certain concentration threshold.

It is also established that elevated ROS results in mitochondrial dysfunction such as change of Mitochondrial Membrane Potential (MMP) and release of apoptosis-inducing factor and cytochrome c.^38–40 To examine these potential mitochondrial dysfunctions, cells were stained with an organic dye, JC-1. The dye molecules are separate at low concentration and emit green fluorescence, but form J-aggregates with red-shifted emission centered around 590 nm. Fig. 3e showed the confocal images of JC-1 stained HepG2 cells after treatment with Qdots. Red JC-1 fluorescence was observed in healthy mitochondria in control HepG2 cells (Fig. 3e), whereas in cells treated with Qdots, green fluorescence was seen, indicating mitochondria membrane damage. Taken all the above molecular characteristics together, it is evident that at high concentration Qdots are capable of inducing apoptosis.

Lastly, we probed the Qdots therapeutic effect under in vivo conditions using a tumour mouse model. One group of mice was given physiologic saline intravenously everyday starting from the day of tumour cell implantation, whereas the other group was injected with Qdots. The inhibition of tumour growth was calculated by surgically excising tumours and measuring the tumour mass. Significantly smaller tumours (0.60 ± 0.17 g) were found in the Qdot treated mice in comparison with the untreated group (1.63 ± 0.15 g), a tumour mass reduction of ∼63.2% (Fig. 4a). The impact of Qdots on survival ability in tumour bearing mice was also evaluated by measuring the survival time. The mean survival time of mice treated with Qdots was increased by 2.5-fold (∼31 days) compared to that of the control group (∼12 days) (Fig. 4b). This result show that the nanotoxicity of Qdots can be potentially converted to a new chemotherapeutic treatment option taking advantage of tumour's enhanced permeability and retention (EPR) and cellular metabolism (cell uptake) effects.

Fig. 4 Antitumour activity of Qdots in vivo. (a) Photographs of tumour were obtained from the treatment and control groups; (b) Kaplan–Meier curves showing survival time of tumour-bearing mice treated with Qdots (red) or physiologic saline (black).

Conclusions

In summary, we demonstrated the possibility of converting Qdots' toxicity, a major concern in molecular imaging, to a traceable chemotherapeutic compound. Using a pair of human liver cells, we show that tumour cell growth is inhibited with an IC₅₀ value in the μM range under in vitro conditions. Under in vivo conditions, the mean survival time of tumour-bearing mice can be extended by 2.5 times when treated with Qdots, owing to the higher binding and uptake of nanoparticles by cancer cells than normal cells. Further development of this technology by fine-tuning the nanoparticle chemical composition, surface coating, size, targeting ligand, and degradation rate can potentially lead to the development of a new treatment (or theranostics due to Qdots' intrinsic fluorescence) option for cancer.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (U1204201, 21501044), and Department of Education Science and Technology Research Key Projects of Henan (13A150063, 16A150004). We are also grateful to Junwei Li and Prof. Xiaohu Gao at the University of Washington for comments and manuscript editing.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures; ¹H NMR spectrum of compound; analysis of ROS by flow cytometry. See DOI: 10.1039/c6ra24063a

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