Rumei Chenga,
Shengju Oub,
Yexu Bua,
Xuan Lia,
Xiaohong Liua,
Yuqin Wanga,
Rui Guoa,
Bingyang Shic,
Dayong Jin*cd and
Yong Liu*ac
aInstitute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China. E-mail: yongliu1980@hotmail.com
bNanjing Landa Femtosecond Insepection Techonlogy Co. Ltd., Nanjing High-tech Industry Development Zone, Nanjing, Jiangsu 210032, China
cARC Center of Excellence for Nanoscale BioPhotonics, Department of Chemistry and Biomolecular Science, Macquarie University, Sydney, NSW 2109, Australia
dInstitute for Biomedical Materials and Devices, Faculty of Science, University of Technology Sydney, NSW 2007, Australia. E-mail: dayong.jin@uts.edu.au
First published on 29th October 2015
We synthesized novel borate antitumor drugs sourced from starch–borate–graphene oxide (SBG) nanocomposites. In vitro results suggest that SBG from the molar ratio of nstarch:
nborate
:
nGO at 2
:
1
:
1 exhibits excellent biocompatibility with normal human cells (>90% cell viability), but are highly toxic against cancer cells (<20% cell viability).
The new-generation carbon nanomaterials of graphene and its derivative e.g. graphene oxide (GO) have recently attracted ever increasing interests both for its implications in fundamental science and for potential numerous applications due to their unique nanoscale structures and physicochemical properties.16,17 Of particular interest to cross-discipline researchers is the interaction between the graphene-based nanomaterials and biomolecules in the areas of cell biology systems.18 Numerous studies have confirmed that the graphene-based scaffolds were compatible with live cells by supporting both their growth and their adhesive properties.19,20 The GO–doxorubicin hydrochloride hybrid was reported as the effective drug delivery system.21 The PEGylate–GO composite exhibited efficient delivery of camptothecin to colon cancer cells.22 Incorporation of GO with polymer based drug has been found to significantly improve the tumor passive uptake of the polymer complexes via enhanced permeability and retention (EPR) effects, which allows effective preferential transport of therapeutic agents to tumor sites via its carbon base.23 Our previous work reported a highly pH-sensitive antitumor drug based on the GO modified chitosan-xanthone Schiff base.24
In this work, we incorporated a unique 2-dimensional nanosheet graphene oxide (GO) to carry the starch–borate complex for enhanced delivery and loading efficiency of the starch–borate complex. We developed a novel structure by covalently linking the oxygen-containing functional groups of GO with starch–borate, and designed the novel nanoscale antitumor drugs with strong toxicity against cancer cells and good biocompatibility with normal cells simultaneously. The concept presented here provides a facile and efficient way to combine the advantages of both the biologic effects of starch and borate and the 2D effect and strong penetrability ability of GO. The resulting SBG shows good biocompatibility to human normal retinal pigment epithelia (RPE) cells while possesses strong cytotoxicity against human CM cells. This is strongly suggestive of a new class of highly efficient targeting antitumor drugs.
The transmission electron microscopy (TEM) image in Fig. 1(b) shows the typical flake-like shape of the resulting SBG and nearly transparent nanosheets as commonly seen in graphene based nanocomposites.25 The thickness of the GO was determined by atomic force microscopy and found to be around 0.7 nm (Fig. 1(c)), suggesting that one layered graphene oxide sheet was prepared. The thickness of the as-synthesized SBG increased to 1.46 nm after incorporation of SB with GO (Fig. 1(d)).
To confirm the successful synthesis of SBG, a Fourier transform infrared spectroscopy (FTIR) technique was used to characterize the functionalized groups of the resulting composite. Fig. 2(a) shows that the FT-IR spectrum of GO (curve i, Fig. 2(a)) records a very simple curve with aromatic C–C stretch (in ring) at 1455 and 1340 cm−1, and a weak CO stretch at 1730 cm−1. As soon as the spectrum of SBG (curve iv, Fig. 2(a)) is concerned, the peak of O–H at 3400 cm−1 becomes broader and stronger. A peak observed at 1646 cm−1 is assigned to hydroxyl group (–OH) bent vibration, indicating that starch complex was bond with GO via the borate mediated conjugating chemistry. The hydrogen bond interaction also existed between different molecular chains.26 Peaks at 1060 cm−1, 1379 cm−1, 1724 cm−1 can be attributed to C–O, O–H, and C
O from carboxylic acid and carbonyl moieties, respectively. The peak at 930 cm−1 from the spectrum of SBG may result from the incorporation of starch with GO.27 SBG nanocomposite was further characterised by X-ray photoelectron spectroscopy (XPS) analysis (Fig. 2(b)). The C1s peak of SBG was observed at 286.2 eV, and differed to that of GO at 285.8 eV.28 The boron peak at 192 eV was evident in the spectrum of SBG while no such peak was observed in GO.29 Both FT-IR and XPS confirm the presence of oxygen functional groups on the GO surface and the successful synthesis of SBG.
Structures of SBG nanocomposites were further measured by Raman spectroscopy. As shown in Fig. 2(c), two characteristic peaks at 1340 cm−1 (D band) and 1582 cm−1 (G band) due to presence of GO are identified, respectively.30 No significant shift is observed when GO is transformed to SBG. However, the intensity ratio of D band to G band (ID/IG), decreased from 0.96 (for GO) to 0.88 (for SBG), suggesting improved integrity and less defects obtained by the introduction of starch into the GO.31 We further performed thermogravimetric analysis (TGA) on the resulting materials. TGA is a very useful technique for identifying different components of a composite.32 As can be seen in Fig. 2(d), the weight loss for all four samples (GO, starch, starch–borate complex, and SBG) in the 50–160 °C range is attributed to the release of adsorbed water. Compared with the other three samples, SBG shows a relatively lower decomposition rate within 300 °C (curve iv, Fig. 2(d)) because of the crosslinking of borate with starch and GO.33 However, the less thermal stability of SBG than starch–borate complex (curve iii, Fig. 2(d)) and the pristine GO (curve ii, Fig. 2(d)) is visible when the temperature is above 300 °C. This may be associated with release of borate from SBG, resulting in a fast decomposition of the isolated starch.
Cytotoxicity of SBG compared with starch–borate and GO with normal ARPE-19 cells and tumorous OCM-1 cells were evaluated using the Cell Counting Kit-8 (CCK-8) assay.34 All samples were sterilized using anhydrous ethanol for 24 h, prior to the co-culturing with two types of cells. Concentration of each sample was remained at 50 μg mL−1. The cell viability of ARPE-19 cells is shown in Fig. 3(a). Most samples (S1, S4, S5) showed the good cell viability which was higher than 70% within a 48 h culture, indicating good biocompatibility of the resulting SBG with normal cells. S4 nanocomposite (nstarch:
nborate
:
nGO = 2
:
1
:
1) exhibited much higher cell viability compared to the other samples for different incubation times. Low cytotoxicity of GO is also observed, well consistent with our previous studies.35 The pristine borate, however, shows a slightly high cytotoxicity. It can be seen in Fig. 3(a) that cytotoxicity of the resulting SBG is dependent on the amount of borate present. The cell viability decreased from 90% to 60% in 48 h when the concentration of borate increased from 25% (S4) to 50% (S2). Sample S3 possesses the highest amount of borate and thus exhibits the lowest ARPE-19 cell viability as shown in Fig. 3(a). As expected, cell viability became enhanced with increasing amounts of starch due to the excellent biocompatibility of starch. Particularly, the sample S5 with highest content of starch (60%) shows a slightly lower cell viability than sample S4 (50%). This is probably attributed to the relative higher percentage of GO at the sample S4 (25%) than that at the sample S5 (20%), confirming the outstanding biocompatibility properties of GO. Since borate is highly toxic to both types of human cells, it is generally not suitable as an antitumor drug which requires high toxicity against tumor cells and good biocompatibility with normal cells. Our present work demonstrates that the borate sandwiched by unique starch and graphene oxide can offer improved biocompatibility with normal cells and targeted suppressive ability against tumor cells. As shown in Fig. 3(b), OCM-1 cell viability with all samples (S1–S5) is less than 30% after 48 h incubation, suggesting good antitumor activity of the resulting SBG. Three samples (S1, S4, S5) which possess good biocompatibility with ARPE-19 cells exhibit good inhibition ability against OCM-1 cells. Of particular interest, the sample S4 shows excellent biocompatibility with ARPE-19 cells (90% cell survival rate) while remaining a superb antitumor ability with OCM-1 cells (20% cell viability), suggesting that as a promising antitumor drug can be developed based on the findings of the sample S4 (i.e. nstarch
:
nborate
:
nGO = 2
:
1
:
1).
For better understanding the antitumor performance of the sample S4, we further performed dose-dependent antitumor study with OCM-1 cells using the sample S4. Concentrations of S4 were varied from 1 to 100 μg mL−1. Fig. 4 shows the OCM-1 cell viability incubated with different amounts of S4 after 24 h and 48 h. It is found that the antitumor ability of S4 exhibits a significant dose-dependent trend. More than 70% tumor cells exhibited a strong suppression rate after 48 h incubation when over 25 μg mL−1 SBG (at the ratio according to S4) was introduced, confirming the outstanding antitumor ability of the resulting SBG.
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Fig. 4 Cell viability of human tumorous OCM-1 cells incubated with different amounts of SBG prepared from nstarch![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
Footnote |
† Electronic supplementary information (ESI) available: Experimental section, TEM, cell viability. See DOI: 10.1039/c5ra17622k |
This journal is © The Royal Society of Chemistry 2015 |