Glioma cell-targeting doxorubicin delivery and redox-responsive release using angiopep-2 decorated carbonaceous nanodots

Abstract

Angiopep-2 modified and doxorubicin loaded carbonaceous nanodots (AN-PEG-DOX-CDs) were synthesized for glioma cell targeting, delivery and redox-responsive release of doxorubicin. Our results provided the possibility of using CDs to construct smart drug delivery systems.

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Designing smart drug delivery systems (DDSs) has emerged as one of the most important strategies for targeted drug delivery and controlled release to improve therapeutic efficiency.^1–4 Various DDSs have been developed with properties that can respond to environmental stimuli, including temperature, proteinases and redox processes.^5–8 Among these, redox-responsive DDSs have gained much attention because this kind of DDS could specifically release drugs into cells, based on the glutathione (GSH) concentration difference between the exterior (2 μM) and interior (10 mM) of cells.⁹ To achieve redox-responsive drug release, a disulfide bond was often used because it could be rapidly cleaved through thiol–disulfide exchange reactions under a reductive environment.^10,11 Therefore, a disulfide bond was used to construct a redox-sensitive DDS in this study.

Carbonaceous nanodots (CDs) have emerged as a novel kind of nanomaterial and possess many functional groups on their surface that make them easy to modify.¹² Thus, the CDs were synthesized as carriers for redox-responsive drug delivery (Fig. 1A). Firstly, amino biocompatible polyethylene glycol (NH₂–PEG) was anchored on the CDs to render high aqueous solubility and stability in physiological solutions, because PEGylation could decrease opsonin adsorption and RES recognition.^13,14 Secondly, angiopep-2, a specific ligand of low density lipoprotein receptor-related protein-1 (LRP1), was modified onto the CDs to mediate the DDS targeting to glioma cells because glioma cells highly express LRP1.^15–17 Thirdly, doxorubicin (DOX), which showed excellent anticancer activities at low concentration,^18,19 was anchored onto the CDs though a linker (cystine) in this study. Therefore, angiopep-2 modified and DOX loaded CDs (AN-PEG-DOX-CDs) were constructed as a kind of redox-responsive DDS to target glioma cells and several experiments were carried out to evaluate their delivery efficiency.

Fig. 1 Characterization of the DOX loaded CDs: (A) a schematic of the AN-PEG-DOX-CDs. (B) UV-vis absorption spectra of DOX, CDs, DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs in water. The DOX absorbance at 480 nm was used to determine the loading efficiency. (C) Fluorescence spectra of DOX, CDs, DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs. Significant fluorescence quenching was observed for DOX loaded on CDs.

The DOX-CDs (DOX loaded CDs), PEG-DOX-CDs (PEG modified DOX-CDs) and AN-PEG-DOX-CDs (angiopep-2 modified PEG-DOX-CDs) were synthesized according to our previous protocol for further experiments.²⁰ The hydrated particle size of the DOX-CDs was 5.5 nm, while the PEG-DOX-CDs and AN-PEG-DOX-CDs presented with larger sizes (6.4 nm and 9.4 nm respectively) than the bare CDs (Fig. 2), which was in accordance with our previous study.²¹ The larger size of the PEG-DOX-CDs and AN-PEG-DOX-CDs may have been due to the PEG (MW = 5000) and angiopep-2 modifications.²⁴

Fig. 2 The size distributions of DOX-CDs (A), PEG-DOX-CDs (B) and AN-PEG-DOX-CDs (C).

As DOX may cause side effects, especially cardiac toxicity, the DOX-loaded DDSs are expected to be stable in blood circulation and release the drugs rapidly after internalization into the tumor cells. To evaluate the redox-sensitive DOX release, we investigated the in vitro release of DOX from the CDs with and without GSH. The fluorescence of DOX was quenched when it was attached to the CDs (Fig. 1C), and the fluorescence of DOX was partly recovered when it was dissociated from the CDs (Fig. S1†), which was based on the nano-surface energy transfer (NSET) effect.^18,22 Thus, the in vitro release of DOX could be well observed via the fluorescence signals of DOX. After 48 h incubation with GSH (10 mM), all the DOX-loaded CDs achieved sustained release behaviors. The release ratios of the DOX-CDs group, PEG-DOX-CDs group and AN-PEG-DOX-CDs group were 94.59%, 83.39% and 77.20%, respectively, and none of the three kinds of DOX-loaded CDs presented an initial burst release (Fig. 3). On the other hand, the 48 h-accumulated DOX release without GSH was only 32.0%, 27.2% and 29.3% for DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs, respectively. The results indicated that the disulfide bond linkages in the DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs can be cleaved under the reduction stimulus of GSH, resulting in the redox-responsive rapid release of DOX, which was useful for delivering DOX specifically into tumor cells. In addition, we compared the in vitro release of the DOX-CDs and DOX-PEG-CDs (DOX was anchored onto the CDs through NH₂–PEG–COOH which didn’t include a disulfide bond). As shown in Fig. S2,† the DOX-PEG-CDs group presented a lower release ratio under the condition of 10 mM GSH, which indicated that the DOX-CDs group presented a good redox-responsive release efficiency compared with the DOX-PEG-CDs group which didn’t include a disulfide bond.

Fig. 3 The in vitro release profiles of the DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs with and without GSH (10 mM) (n = 3, mean ± SD).

In order to determine the glioma cell targeting efficiency of these DOX-tethered CDs, the cellular uptake of the DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs (with a DOX concentration of 5.2 μg mL⁻¹) by C6 glioma cells was investigated using a confocal laser scanning microscope. As shown in Fig. 4, the DOX fluorescence in the cells treated with the DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs was mainly in the cytoplasm after 1 h of incubation. When the incubation time was extended to 4 h, the DOX fluorescence of all the groups was mainly located in the nucleus. It demonstrated that the higher intracellular GSH concentration results in a rapid DOX release in the cytoplasm, owing to the disulfide bond cleavage after 1 h of incubation, and the released DOX could mainly be located in the nucleus after 4 h. What’s more, the DOX-CDs and PEG-DOX-CDs groups presented with similar uptake intensities, while the uptake intensity of the AN-PEG-DOX-CDs group was higher than those of the DOX-CDs and PEG-DOX-CDs groups, both at 1 h and 4 h, indicating the targeting efficiency of the angiopep-2 on the C6 cells.¹⁹ In addition, we investigated the cellular uptake of the DOX-CDs group and DOX-PEG-CDs group (with DOX concentrations of 15 μg mL⁻¹). As shown in Fig. S3,† the DOX-CDs group presented a higher cellular uptake than the DOX-PEG-CDs group, both at 1 h and 4 h, demonstrating the redox-responsive release behavior of the DOX-CDs.

Fig. 4 Confocal microscopy images of the cellular uptake of the DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs into C6 cells after incubations of 1 h and 4 h. Blue represents the nuclei, green represents DOX and the bar represents 50 μm.

The cellular uptake of the DOX-tethered CDs was further evaluated with C6 cells using flow cytometry. After the DOX-tethered CDs (with DOX concentrations of 5.2 μg mL⁻¹) were added to the culture medium, the C6 cells were cultured under 37 °C for 1 h and 4 h. As shown in Fig. 5, the fluorescence intensity between the DOX-CDs group and the PEG-DOX-CDs group presented no significant difference at 1 h, while the fluorescence intensity of the AN-PEG-DOX-CDs group was 2.4-fold and 2.6-fold higher than that of the DOX-CDs group and PEG-DOX-CDs group, respectively, at 1 h. A similar tendency was observed when the incubation time was extended to 4 h. These data demonstrated that angiopep-2 could specifically recognize the highly expressed LRP1 on C6 cells, thus facilitating the internalization of the AN-PEG-DOX-CDs.²³

Fig. 5 The cellular uptake of the DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs on C6 cells measured using a flow cytometer (n = 3, mean ± SD). *, ** and *** demonstrate p < 0.05, p < 0.01 and p < 0.001, respectively, between the compared groups; n.s means no significant difference.

To evaluate the therapeutic efficiency of the DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs, an MTT assay on C6 cells was carried out. The results showed that the DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs all afforded high anticancer activities in vitro on C6 cells (Fig. 6). Besides, when the concentration of DOX reached 7.35 μg mL⁻¹, the DOX-CDs, PEG-DOX-CDs and AN-PEG-DOX-CDs could cause about 50% cell growth inhibition, which showed a similar efficiency to the free DOX, indicating that the DOX loaded CDs presented a complete release of DOX after 24 h incubation. Interestingly, the AN-PEG-DOX-CDs group showed a slightly higher inhibition rate than the free DOX at the higher concentrations (7.35 μg mL⁻¹ and 1.47 μg mL⁻¹) because of their enhanced cellular uptake. Besides, the free CDs show low cytotoxicity at a similar concentration according to our previous study.²⁴ This high antitumor activity of the DOX-tethered CDs may be due to the efficient cellular uptake abilities mentioned above and the quick release of DOX owing to the cleavage of the disulfide bonds triggered by the high GSH concentration in the cytoplasm after the cellular uptake, which is consistent with other studies.⁵

Fig. 6 In vitro cell toxicity assay on C6 cells (n = 3, mean ± SD).

In conclusion, AN-PEG-DOX-CDs were prepared with abilities of redox-responsive drug release and glioma cell targeting. The in vitro drug release results demonstrate that the AN-PEG-DOX-CDs presented a higher DOX release efficiency under the reduction stimulus of GSH (10 mM) than the groups without GSH. In addition, confocal microscopy and flow cytometry analyses indicate that the AN-PEG-DOX-CDs showed high uptake efficiency owing to the angiopep-2 modification. What’s more, the cytotoxicity of the DOX-loaded CDs was investigated, and the AN-PEG-DOX-CDs appeared highly efficient at cellular proliferation inhibition as well as the free DOX, owing to the redox-responsive DOX release under the GSH stimulus and the targeting efficiency of angiopep-2. In summary, the redox-responsive AN-PEG-DOX-CDs could open up a window to the potential use of CDs as drug carriers for constructing novel drug delivery systems.

Acknowledgements

The work was granted by National Natural Science Foundation of China (81402866) and the National Basic Research Program of China (973 Program, 2013CB932504).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra08245e

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