Nano graphene oxide–hyaluronic acid conjugate for target specific cancer drug delivery

Abstract

A nano graphene oxide–hyaluronic acid (NGO–HA) conjugate was successfully prepared for target specific delivery of an anti-cancer drug loaded by π–π stacking via HA receptor mediated endocytosis. In vitro tests confirmed the pH dependent drug release and target specific anti-cancer effect of the complex.

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Graphene oxide (GO) is an oxidized form of graphene which can be easily functionalized on the sp² domain with hydroxyl, epoxy, and carboxyl groups.^1,2 Nano-sized GO (NGO) has a high potential to be used as a drug carrier due to the fact that both sides of the graphitic domain are available to load a variety of drugs by π–π stacking, hydrophobic interaction, and hydrogen bonding.^3–5 In addition, NGO can be used as a photo-absorber for a photothermal ablation therapy.⁶ However, the poor stability of NGO should be circumvented for the application as a drug carrier in serum and high ionic strength solution.⁷ The stabilized NGO has been used for the delivery of anti-cancer drug,⁸ siRNA,⁹ and photosensitizer.¹⁰ Pan et al. developed NGO modified with poly(N-isopropylacrylamide) as a drug carrier of camptothecin, which showed anti-proliferation effect on A-5RT3 cancer cells.¹¹ Zhang et al. tethered folic acid as a targeting moiety and poly(ethylene glycol), PEG, as a stabilizer on NGO for target specific treatment of MCF-7 cancer cells.¹² Instead of such a multiple introduction of functional groups on NGO, we carried out the conjugation of NGO with a biopolymer of hyaluronic acid (HA) for target specific treatment of cancer cells.

HA is a naturally occurring linear polysaccharide and has been considered as one of the best biopolymers in terms of safety issues. HA has been widely used for drug delivery systems,^13,14 regenerative medicines,¹⁵ and other biomedical applications. HA is easy to be chemically modified for the preparation of HA derivatives with functional groups.¹⁶ Especially, HA has been utilized for long acting and target specific delivery of biopharmaceuticals on the basis of the negligible nonspecific interaction with serum proteins¹⁷ and effective tissue targeting through receptors such as cluster determinant 44 (CD44),¹⁸ hyaluronan receptor for endocytosis (HARE),^19,20 and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1).²¹ HA degrades into short saccharide units after the receptor mediated internalization, which is the critical role of HA catabolism.²² We previously reported long acting and target specific HA–interferon α conjugates for the treatment of hepatitis C virus infection¹⁴ and layer-by-layer assembled AuNP/siRNA/PEI/HA nano-complexes for target specific gene silencing in the liver.¹⁷

In this work, a target specific drug delivery system using NGO–HA conjugate was developed for the treatment of cancer. Epirubicin, which is a chemical cancer drug favoured over doxorubicin with a relatively low side effect, was loaded on the surface of NGO–HA by π–π stacking. Fig. 1 shows a schematic illustration for the target specific cancer drug delivery system of epirubicin/NGO–HA complex. After bioimaging for the drug release profile by confocal laser scanning microscopy (CLSM), in vitro tests were carried out to confirm the pH dependent drug release and target specific anti-cancer effect of the complex.

Fig. 1 Schematic illustration for the preparation process of epirubicin/NGO–HA complex and the target specific delivery of the complex to cancer cell via HA receptor mediated endocytosis.

NGO–HA was prepared by conjugation of carboxylated NGO with 38% hexamethylenediamine modified HA (HA–HMDA, ESI Fig. S1†), as we reported elsewhere.⁶ TEM image of NGO–HA revealed a spherical morphology of NGO–HA nanoparticles with a mean particle size of ca. 250 nm (Fig. 2a). After preparation of NGO–HA, epirubicin was loaded on NGO–HA at pH 9 and 4 °C for 24 h. Epirubicin/NGO–HA complex was filtered to remove the remaining epirubicin with a 300 kDa centrifugal filter three times. The amount of loaded epirubicin was measured using a standard curve obtained with a UV-Vis spectrometer at 490 nm (Fig. S2†). The UV-Vis absorbance spectrum of NGO showed a π → π transition absorbance peak at 230 nm. In contrast, epirubicin/NGO–HA complex had two peaks at 230 nm and 490 nm confirming the successful loading of epirubicin on the NGO–HA (Fig. S2†). The loading amount of epirubicin on NGO–HA was varied depending on the pH of epirubicin solutions. When the initial concentration of epirubicin was 0.27 mg mL⁻¹ (0.1 mM), the loading amount (%) of epirubicin on NGO–HA was 2% at pH 4, 9% at pH 7 and 25% at pH 9. The pH dependent loading characteristics of epirubicin onto NGO–HA might be advantageous for the intracellular delivery of cancer drugs, considering the acidic environment of endosomes. For further applications, the colloidal stability of epirubicin/NGO–HA complex was tested in 100 mM of NaCl solution. In comparison to NGO and NGO–COOH, NGO–HA showed good physiological stability in the NaCl solution before and after epirubicin loading (Fig. S3† and 2b). The NGO–HA solution remained stable for several months without aggregation. The hydrodynamic size of NGO–HA conjugate was ca. 400 nm according to DLS analysis (Fig. 2c), which was relatively larger than that by TEM in a dried state. Fig. 2d shows the MTT assay results after incubation of B16F1 melanoma cells with NGO–HA at various concentrations for 24 h. B16F1 cells have HA receptors such as CD44 and LYVE-1.¹⁶ NGO–HA showed no significant toxicity up to the concentration of 500 μg mL⁻¹.

Fig. 2 (a) TEM image of NGO–HA nanoparticle (scale bar = 500 nm). (b) The colloidal stability of NGO, NGO–COOH and NGO–HA after epirubicin loading. (c) DLS analysis of NGO and NGO–HA. (d) Relative cell viability after incubation of B16F1 cells with the drug carrier of NGO–HA conjugate.

To investigate the HA receptor mediated endocytosis of the epirubicin/NGO–HA complex, CLSM analysis was carried out after incubation of B16F1 melanoma cells with the complex in the presence of green fluorescence lysotracker. When B16F1 cells were incubated with epirubicin, red fluorescence was localized in nucleus and green fluorescence was distributed in entire cytosol indicating the direct diffusion of epirubicin into nucleus (Fig. 3a and b). After incubation of B16F1 cells with epirubicin/NGO–HA complex, interestingly, we could observe a yellow fluorescence by co-localization of the complex and the lysotracker in cytosol (Fig. 3c). When the cells were treated with epirubicin/NGO–HA complex in the presence of free HA, green lysotracker signal was dominant due to the blocking of cell membranes by free HA (Fig. 3d). The concentration of epirubicin and epirubicin/NGO–HA was fixed at 20 μg mL⁻¹. The co-localization of epirubicin/NGO–HA complex and lysotracker confirmed the HA receptor mediated endocytosis of epirubicin/NGO–HA complex into the cancer cells.

Fig. 3 Confocal laser scanning microscopic images of B16F1 cells after incubation with (a and b) epirubicin and (c and d) epirubicin/NGO–HA complex in (a and c) the absence and (b and d) the presence of free HA molecules. The cytosol of B16F1 cells was stained with green fluorescence lysotracker.

The time dependent epirubicin release from epirubicin/NGO–HA complex into cell nucleus was monitored after incubation with the sample at an epirubicin concentration of 20 μg mL⁻¹ for 10 min, 1 h, 12 h, and 24 h. In case of the treatment with free epirubicin, epirubicin was rapidly delivered to the nucleus (Fig. 4a and b) and then dispersed in the cytosol with increasing time (Fig. 4c and d), likely due to the cell death. In contrast, epirubicin/NGO–HA complex was delivered to the cytosol by the HA receptor mediated endocytosis and the loaded epirubicin was released from the complex to the nucleus with increasing time (Fig. 4e–h). The release of epirubicin from the complex might be facilitated in the endosome at a low pH around 5.

Fig. 4 Confocal laser scanning microscopic images of B16F1 cells showing the different drug delivery patterns after treatment with epirubicin for (a) 10 min, (b) 1 h, (c) 12 h, (d) 24 h and with epirubicin/NGO–HA complex for (e) 10 min, (f) 1 h, (g) 12 h, (h) 24 h, respectively (scale bar = 20 μm).

Fig. 5a shows in vitro release profiles of epirubicin from the epirubicin/NGO–HA complex in PBS at pH 5 and 7. Within the first 24 h, 50% of epirubicin was released from the complex at pH 5 and 70% of epirubicin was released in 72 h, whereas only 18% of epirubicin was released from the complex at pH 7 in 72 h. The enhanced release of epirubicin at low pH confirmed the feasibility of epirubicin/NGO–HA complex for the intracellular delivery of cancer drugs.²³ The anti-tumor effect of the released epirubicin was assessed by in vitro cell viability tests in B16F1 cells with HA receptors such as CD44 and LYVE-1. At the NGO–HA concentration of 50 μg mL⁻¹, we assessed the anti-tumor effect of epirubicin/NGO–HA complex up to 100 μM of the epirubicin concentration, which resulted in the effective inhibition of tumor growth after incubation for 24 h (ESI Fig. S4†).

Fig. 5 (a) In vitro release profiles of epirubicin from the epirubicin/NGO–HA complex at pH 5 and pH 7. (b) Relative cell viability after treatment of B16F1 cells with epirubicin and epirubicin/NGO–HA complex for 30 min in the absence and presence of free HA. The results represent mean ± standard deviation (n = 3, **P < 0.01).

The target specific anti-tumour effect of the epirubicin/NGO–HA complex was investigated in B16F1 cells by MTT assay with and without HA pre-incubation. As shown in Fig. 5b, the treatment of B16F1 cells with epirubicin/NGO–HA complex in the presence of free HA for 30 min resulted in 40% higher cell viability at an epirubicin concentration of 20 μg mL⁻¹ than that in the absence of HA. In the early stage of incubation (30 min), epirubicin/NGO–HA complex was more rapidly delivered to B16F1 cells by HA receptor mediated endocytosis, showing statistically higher anti-tumor effect than free epirubicin. There was no big difference in cell viability for the case of epirubicin treatment with and without HA pre-incubation. The blocking of HA receptors in the presence of an excess amount of HA might inhibit the receptor mediated endocytosis of the epirubicin/NGO–HA complex. All these results were well matched with the confocal microscopic analysis. Furthermore, we could confirm the statistically higher anti-tumour effect of epirubicin/NGO–HA complex than free epirubicin in various cancer cells expressing HA receptors on their surfaces including MCF-7, MDA-MB-231, and HeLa cells (Fig. S5†). The epirubicin/NGO–HA complex might be applied for a combination therapy such as chemo- and photo-therapy using a NIR laser. The drug carrier of NGO–HA conjugate can be also used to load multiple chemical drugs by π–π stacking, hydrogen bonding, and hydrophobic interaction for the target specific and simultaneous treatment of various diseases.

Conclusions

Epirubicin/NGO–HA complex was successfully developed for the target specific treatment of cancer cells. With enhanced serum stability, the graphitic domain of NGO in NGO–HA could be exploited to load a model chemical cancer drug of epirubicin by π–π interaction. Confocal microscopy clearly visualized the co-localization of epirubicin/NGO–HA complex and the lysotracker, reflecting the HA receptor mediated endocytosis. In vitro release tests revealed the pH dependent loading and releasing behavior of epirubicin. The anti-tumor effect of released epirubicin was confirmed in B16F1 cells by MTT assay. The enhanced release of epirubicin at an acidic condition might be beneficial for the target specific intracellular delivery of cancer drugs. Taken together, we could confirm the feasibility of epirubicin/NGO–HA complex for the treatment of various diseases related with HA receptors.

Acknowledgements

This work was financially supported by the Converging Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0081871). This study was also supported by Mid-career Researcher Program through NRF grant funded by the MEST (no. 2012R1A2 A2A06045773).

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Footnote

† Electronic supplementary information (ESI) available: Materials and methods, and experimental procedures. See DOI: 10.1039/c4ra00605d

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