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A small-sized graphene oxide supramolecular assembly for targeted delivery of camptothecin† Ying-Ming Zhang,a Yu Cao,a Yang Yang,a Jia-Tong Chenb and Yu Liu*a
Received 15th June 2014, Accepted 5th September 2014 DOI: 10.1039/c4cc04533e www.rsc.org/chemcomm
A small-sized graphene oxide supramolecular assembly was obtained by the inclusion complexation of hyaluronated adamantane with b-cyclodextrin and the p-stacking of graphene oxide with camptothecin, exhibiting an excellent stability in the serum environment and a higher inhibition effect toward malignant cells than a free drug.
Although there is a growing consensus that many hydrophobic drugs and biologically active molecules have great therapeutic potential, the poor aqueous solubility, nonspecific uptake, and rapid clearance in the bloodstream severely impede their development from the fundamental study to the clinical use.1 To this end, the construction of carrier-mediated artificial systems through a supramolecular methodology offers an alternative and even a more powerful strategy to address or overcome these important barriers to drug formulation and delivery.2,3 In this regard, the combination of cell-specific targeting ligands with macrocyclic receptors may confer several practical superiorities in the context of medical and gene therapies,4 because the representative macrocycles, such as cyclodextrin (CD)5 and cucurbituril,6 can provide a modifiable structure and a hydrophobic microenvironment to simultaneously incorporate chemical and biological functionalities into the supramolecular assembled entities, thus leading to the enhancement in drug bioavailability with minimal long-term side effects. Nevertheless, the more efficient macrocycle-based delivery systems with suitable size, synergistic therapeutic effect and good stability in physiological environments still deserve our careful attention. Here, we report a conjugated delivery system capable of binding the targeting agents and drug molecules by the noncovalent multiple interactions with b-CD functionalized graphene oxide (GO-CD) as scaffolds (Scheme 1). There are some inherent advantages of such a system. Firstly, among the three main components of this system, a
Department of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, P. R. China. E-mail: [email protected] b Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, P. R. China † Electronic supplementary information (ESI) available: Experimental details and additional figures. See DOI: 10.1039/c4cc04533e
13066 | Chem. Commun., 2014, 50, 13066--13069
Scheme 1
Construction of CPT@GO-CD–HA-ADA supramolecular assembly.
CD, a class of cyclic oligosaccharides, and hyaluronic acid, a widely distributed glycosaminoglycan, are all biocompatible, while the pristine GO-CD could be disrupted from several hundreds to tens of nanometres, and the resultant uniform and small-sized GO nanosheets may facilitate its biodegradation in the intracellular and extracellular environments.7 Secondly, the targeting ability derives from the hyaluronated adamantane (HA-ADA) chains, in which the HA skeleton can specifically recognize the HA receptorexpressing tumor cells in cancer metastasis (Fig. S1 in the ESI†).8 Thirdly, anticancer drugs, such as camptothecin (CPT), could be readily integrated into the water-soluble nanocarrier of GO-CD–HAADA (Scheme S1, ESI†). Consequently, this highly stable and compatible nanostructure is considered as an effective therapeutic for oncological treatment. The chemical structure and synthetic route for the preparation of the CPT@GO-CD–HA-ADA conjugate are shown in Scheme 1. For a biocompatible purpose, the size of GO sheets was controlled around 300–500 nm by simply adjusting the oxidation conditions
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and the reaction time during the chemical exfoliation process.9 Subsequently, the GO nanosheets were functionalized by grafting b-CD units via an amine-epoxy reaction.10 Moreover, benefiting from a two-step orthogonal noncovalent interaction, i.e., the supramolecular complexation of b-CD cavity with the adamantyl group and the p–p stacking interaction between the planar GO surface and the aromatic ring of the drug molecule, CPT, a cytotoxic quinoline alkaloid in cancer chemotherapy, was successfully endowed with water solubility and the targeting ability to bind malignant cells. The qualitative characterization for the formation of GO-CD–HAADA complex began with UV/vis, FTIR, and TGA experiments. In comparison to free GO, the absorption of GO-CD resulted in an obvious bathochromic shift from 227 to 256 nm, indicating the recovery of coplanarity to some extent after the removal of epoxy groups on GO sheets.11 In addition, no spectral change was observed upon complexation with HA-ADA, implying that the introduction of optically transparent HA-ADA component cannot affect the integrity of the GO-CD platform (Fig. S2 in the ESI†). Accordingly, some characteristic vibrations at 2928, 1155, 1078, and 1653 cm 1 in the FTIR spectrum of GO-CD–HA-ADA, corresponding to C–H and C–O in CD units and CO–NH in the HA-ADA polymer, respectively, jointly confirm the noncovalent modification of graphene sheets with the HA polymer by inclusion complexation (Fig. S3 in the ESI†). Furthermore, as can be seen from Fig. S4 (ESI†), in spite of a similar change profile, the decomposition temperatures of GO-CD–HA-ADA always situate themselves between the ones of GO-CD and HA-ADA in the region from room temperature to 800 1C, suggesting that the binary complex maintained its thermal stability compared to its precursors. The morphological information of the supramolecular assembly was visually obtained using atomic force microscopy (AFM) and dynamic light scattering (DLS) measurements. As can be seen from Fig. 1a, free GO gave a monodisperse nanosheet with an average height of 1.1 nm, corresponding to the completely exfoliated GO single layer. In addition, the height of GO-CD conjugate increased to 2.5 nm, which was in accordance with the attachment of CD units (height 0.8 nm) onto both sides of the GO surface (Fig. 1b). Moreover, an average height of 4.5 nm with a lateral dimension ranging from 50 to 150 nm in the AFM image of the GO-CD–HA-ADA complex was attributed to the cross-linking of GO-CD with the HA-ADA chain through intermolecular communication (Fig. 1c). In contrast, the cross-section analyses reveal that after loading CPT, the height of the ternary assembly of CPT@GO-CD–HA-ADA was ca. 6.4 nm, larger than that of GO, GO-CD, and GO-CD–HAADA, which would be originated from the spatial arrangement of CPT with the monolayer GO-CD and b-CD-appended HA polymer (Fig. 1d). Along with the microscopic investigation results, it is also found that there was an obvious morphological change in aqueous solution; that is, the average hydrodynamic diameter of GO-CD was 400 nm, whereas the one of GO-CD–HA-ADA was decreased to 80 nm at the same concentration, further suggesting that the continuous ultrasonication in the presence of the HA-ADA polymer could crack the GO sheet into small-sized components and then promote the eventual receptor-mediated internalization of the biocompatible supramolecular complex by cells (Fig. S5 in the ESI†).
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Fig. 1 Typical AFM images of (a) GO, (b) GO-CD, (c) GO-CD–HA-ADA, and (d) CPT@GO-CD–HA-ADA, respectively.
Subsequently, quantitative evidence regarding the composition and structure of CPT@GO-CD–HA-ADA was further provided by X-ray photoelectron spectroscopy (XPS). As expected, all the peaks of associated elements were recorded in the XPS survey spectra, where Na+ was introduced as the counterion of carboxylate groups on GO and HA backbones (Fig. 2 and Fig. S6–S7 in ESI†). Taking the CPT@GO-CD–HA-ADA conjugate as an example, the binding energy positions at 285, 398, and 530 eV were assigned to C1s, N1s, and O1s, respectively (Fig. 2c). The detailed XPS analyses are described in
Fig. 2 XPS survey spectra of (a) GO-CD, (b) GO-CD–HA-ADA, and (c) CPT@GO-CD–HA-ADA, respectively.
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the ESI.† Moreover, through a calculation based on the content ratio of nitrogen atoms in GO-CD (0.67%), GO-CD–HA-ADA (2.13%), and CPT@GO-CD–HA-ADA (2.36%), it can be verified that the mass fractions of CD in GO-CD and HA-ADA in GO-CD–HA-ADA were 27.7% and 40.5%, while that of CPT in CPT@GO-CD–HA-ADA was 3.7%, respectively. Therefore, the loading ratio of CPT on the GO surface was obtained as 3.7%, which was in good agreement with the UV/vis spectroscopic titration results as described below. It is significant to note that GO-CD–HA-ADA exhibited an excellent stability in both saline and serum environments, and this biocompatible property can be readily distinguished by the change of solution state after centrifugation. As shown in Fig. S8 (ESI†), GO-CD in phosphate buffer solution (PBS) and bovine serum albumin (BSA) was much less resistant to centrifugal force, as compared to the resultant complex that was stably dispersed under the same experimental conditions. This large disparity in the stability of GO-CD and GO-CD–HA-ADA implies that the formation of inclusion complex can greatly deter the negatively charged GO planes from the undesirable self-associated aggregation in aqueous solution. Consequently, in the CPT loading test, it is found that the solution of CPT@GO-CD could cause a very serious precipitation in PBS; while the one in the presence of HA-ADA still remained homogeneous and clear even after standing for 10 h (Fig. S9 in the ESI†). As a consequence of the favourable p-stacking interaction, CPT can be conveniently introduced onto the GO surface. As expected, a new band at 366 nm was observed in the UV/vis spectrum of CPT@GO-CD–HA-ADA, which is the characteristic absorption of CPT (Fig. 3).12 Meanwhile, the emission intensity of CPT was decreased by 92% after loading onto GO, mainly due to the high fluorescence quenching efficiency of GO sheets (Fig. S10 in the ESI†).13 In our case, the absorption of GO-CD–HA-ADA as the reference could be actually deducted from the UV/vis spectrum of CPT@GO-CD–HA-ADA, and then the net concentrations of CPT and GO-CD–HA-ADA were accordingly calculated using their molar absorption coefficients at 366 and 800 nm, respectively (see the ESI† for details). Therefore, the drug loading efficiency was obtained as
Fig. 3 UV/vis absorption of GO-CD–HA-ADA and CPT@GO-CD–HAADA, respectively ([GO-CD–HA-ADA] = 64.0 mg mL 1). Inset: release profile of CPT from the CPT@GO-CD–HA-ADA conjugate in vitro in PBS (pH 7.2, I = 0.01 M) at 37 1C.
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3.3%, which was quite similar to the XPS analysis results (3.7%). Moreover, the releasing behaviours of CPT from the GO-CD–HA-ADA nanoplatform were examined using fluorescence spectroscopy in PBS at 37 1C, providing a controlled and sustained release of CPT over a period of 10 h (Fig. 3, inset). Comparatively, the rate of drug release was much faster at pH 5.7, and this pH-responsive releasing behaviour could definitely inhibit the growth and reproduction of tumor cells in cancer cell environments (Fig. S11 in the ESI†). To further explore the molecular binding mode of CPT with GO-CD and HA-ADA, some control experiments were performed. As illustrated in Fig. S12 (ESI†), when CPT was mixed with GO-CD, the absorption peak at 366 nm was reproduced, which resembled the one in the CPT@GO-CD–HA-ADA system. Instead, no absorption beyond 300 nm was observed in the case of CPT with HA-ADA, convincingly proving that p-stacking interaction is the primary driving force to trap drug molecules onto the GO surface and the existence of HAADA cannot make any negative impact on the loading ratio of CPT (Fig. S13 in the ESI†). Finally, cytotoxicity experiments were carried out to evaluate the antitumor activity of the ternary assembly in vitro. Gratifyingly, superior to CPT and CPT@GO-CD, CPT@GO-CD–HA-ADA displayed a better anticancer activity toward MDA-MB-231 cancer cells, a type of human breast cancer cells with abundant HA receptors being overexpressed on its surface (Fig. 4a and Fig. S14a in the ESI†).14 After a 48 h incubation, the relative cellular viability of MDA-MB-231 for CPT@GO-CD–HA-ADA was 51.5%, which was lower than the corresponding value for free CPT (64.5%). Significantly, the relative
Fig. 4 Relative cellular viability and cell photos of (a–g) MDA-MB-231 and (h–m) NIH3T3 cell lines after the treatment with blank (b and i), CPT (c and j), GO-CD–HA-ADA (d and k), CPT@GO-CD–HA-ADA (e and l), CPT@GO-CD– HA-ADA with an excess of HA (f), and CPT@GO-CD (g and m), respectively, in 48 h incubation ([CPT] = 1.0 mM). The statistically significant differences were indicated with asterisks (P o 0.05).
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cellular viability of normal fibroblasts NIH3T3 with CPT@GO-CD– HA-ADA was 82.5%, which was quite high compared to that with free CPT (63.0%). Furthermore, when the receptors on the cancer cell surface were blocked by an excess amount of HA, the nanocarrier lost its original cell selectivity, giving a cellular viability that was nearly the same as the value obtained using free drugs. In particular, it is a remarkable fact that neither abnormal apoptosis nor cellular morphological change was observed in the GO-CD–HA-ADA group (Fig. 4d and k). These results jointly demonstrate that the obtained GO-CD–HA-ADA carrier could be utilized as a safe and promising candidate for drug delivery and other biomedical research. In conclusion, a tumor-targeted delivery system for CPT was constructed by the influence of supramolecular positive cooperativity of GO-CD with HA-ADA, displaying a satisfactory stability and biocompatibility under physiological conditions. The CD–ADA inclusion complex can largely prevent the GO skeletons from intermolecular aggregation in water, which then facilitate the disruption of a pristine GO sheet into small-sized components under ultrasonic conditions. Moreover, as investigated using cytotoxicity experiments, it can be seen that the ternary assembly of CPT@GO-CD–HA-ADA exhibited a higher curative effect and a lower cytotoxicity than a free drug. In principle, the simple loading and specific delivery of CPT in this work further stress the importance of facile synergetic interaction in the design and engineering of macrocycle-based drug carriers, by which the conventional chemotherapeutic drugs can be specifically delivered to their intended sites of action and their general toxicity can be reduced to a significant extent for wider clinical application. We thank the 973 Program (2011CB932502) and NNSFC (91227107 and 21102075) for financial support.
Notes and references 1 (a) H.-J. Schneider, P. Agrawal and A. K. Yatsimirsky, Chem. Soc. Rev., 2013, 42, 6777; (b) K. Kawakami, M. Ebara, H. Izawa, N. M. SanchezBallester, J. P. Hill and K. Ariga, Curr. Med. Chem., 2012, 19, 2388;
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13 14
¨nder and K. Landfester, Biomacromolecules, 2009, 10, (c) V. Maila 2379; (d) R. Duncan, Nat. Rev. Cancer, 2006, 6, 688. (a) W. Cao, Y. Gu, M. Meineck and H. Xu, Chem. – Asian J., 2014, ˘, 9, 48; (b) H. M. Aliabadi, B. Landry, C. Sun, T. Tang and H. Uludag Biomaterials, 2012, 33, 2546; (c) E. Gravel, J. Ogier, T. Arnauld, ´ and E. Doris, Chem. – Eur. J., 2012, N. Mackiewicz, F. Duconge 18, 400; (d) L. J. De Cock, S. De Koker, B. G. De Geest, J. Grooten, C. Vervaet, J. P. Remon, G. B. Sukhorukov and M. N. Antipina, Angew. Chem., Int. Ed., 2010, 49, 6954; (e) J. Buse and A. El-Aneed, Nanomedicine, 2010, 5, 1237. (a) L. Chen, J. Wu, C. Schmuck and H. Tian, Chem. Commun., 2014, 50, 6443; (b) H.-L. Zhang, Y. Zang, J. Xie, J. Li, G.-R. Chen, X.-P. He and H. Tian, Sci. Rep., 2014, 4, 5513, DOI: 10.1038/srep05513; (c) X.-P. He, Q. Deng, L. Cai, C.-Z. Wang, Y. Zang, J. Li, G.-R. Chen and H. Tian, ACS Appl. Mater. Interfaces, 2014, 6, 5379. (a) F. Sansone and A. Casnati, Chem. Soc. Rev., 2013, 42, 4623; (b) E. Marsault and M. L. Peterson, J. Med. Chem., 2011, 54, 1961; (c) R. E. Mewis and S. J. Archibald, Coord. Chem. Rev., 2010, 254, 1686; (d) E. M. Driggers, S. P. Hale, J. Lee and N. K. Terrett, Nat. Rev. Drug Discovery, 2008, 7, 608. (a) Y. Chen and Y. Liu, Chem. Soc. Rev., 2010, 39, 495; (b) M. E. Brewster and T. Loftsson, Adv. Drug Delivery Rev., 2007, 59, 645; (c) D. W. Bartlett, H. Su, I. J. Hildebrandt, W. A. Weber and M. E. Davis, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 15549. (a) I. Ghosh and W. M. Nau, Adv. Drug Delivery Rev., 2012, 64, 764; (b) L. Wang, L.-L. Li, Y.-S. Fan and H. Wang, Adv. Mater., 2013, 25, 3888. A. Bianco, Angew. Chem., Int. Ed., 2013, 52, 4986. (a) Y. Yang, Y.-M. Zhang, Y. Chen, J.-T. Chen and Y. Liu, J. Med. Chem., 2013, 56, 9725; (b) N. Li, Y. Chen, Y.-M. Zhang, Y. Yang, Y. Su, J.-T. Chen and Y. Liu, Sci. Rep., 2014, 4, 4164, DOI: 10.1038/ srep04164. L. Zhang, J. Liang, Y. Huang, Y. Ma, Y. Wang and Y. Chen, Carbon, 2009, 47, 3365. J. Liu, G. Chen and M. Jiang, Macromolecules, 2011, 44, 7682. J. Zhang, H. Yang, G. Shen, P. Cheng, J. Zhang and S. Guo, Chem. Commun., 2010, 46, 1112. (a) N. G. Sahoo, H. Bao, Y. Pan, M. Pal, M. Kakran, H. K. F. Cheng, L. Li and L. P. Tan, Chem. Commun., 2011, 47, 5235; (b) L. Zhang, J. Xia, Q. Zhao, L. Liu and Z. Zhang, Small, 2010, 6, 537; (c) Z. Liu, J. T. Robinson, X. Sun and H. Dai, J. Am. Chem. Soc., 2008, 130, 10876. J. Geng and H.-T. Jung, J. Phys. Chem. C, 2010, 114, 8227. (a) M. Kaufmann, K.-H. Heider, H.-P. Sinn, G. von Minckwitz, H. Ponta and P. Herrlich, Lancet, 1995, 345, 615; (b) P. Auvinen, R. Tammi, M. Tammi, R. Johansson and V.-M. Kosma, Histopathology, 2005, 47, 420.
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A small-sized graphene oxide supramolecular assembly for targeted delivery of camptothecin† Ying-Ming Zhang,a Yu Cao,a Yang Yang,a Jia-Tong Chenb and Yu Liu*a
Received 15th June 2014, Accepted 5th September 2014 DOI: 10.1039/c4cc04533e www.rsc.org/chemcomm
A small-sized graphene oxide supramolecular assembly was obtained by the inclusion complexation of hyaluronated adamantane with b-cyclodextrin and the p-stacking of graphene oxide with camptothecin, exhibiting an excellent stability in the serum environment and a higher inhibition effect toward malignant cells than a free drug.
Although there is a growing consensus that many hydrophobic drugs and biologically active molecules have great therapeutic potential, the poor aqueous solubility, nonspecific uptake, and rapid clearance in the bloodstream severely impede their development from the fundamental study to the clinical use.1 To this end, the construction of carrier-mediated artificial systems through a supramolecular methodology offers an alternative and even a more powerful strategy to address or overcome these important barriers to drug formulation and delivery.2,3 In this regard, the combination of cell-specific targeting ligands with macrocyclic receptors may confer several practical superiorities in the context of medical and gene therapies,4 because the representative macrocycles, such as cyclodextrin (CD)5 and cucurbituril,6 can provide a modifiable structure and a hydrophobic microenvironment to simultaneously incorporate chemical and biological functionalities into the supramolecular assembled entities, thus leading to the enhancement in drug bioavailability with minimal long-term side effects. Nevertheless, the more efficient macrocycle-based delivery systems with suitable size, synergistic therapeutic effect and good stability in physiological environments still deserve our careful attention. Here, we report a conjugated delivery system capable of binding the targeting agents and drug molecules by the noncovalent multiple interactions with b-CD functionalized graphene oxide (GO-CD) as scaffolds (Scheme 1). There are some inherent advantages of such a system. Firstly, among the three main components of this system, a
Department of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, P. R. China. E-mail: [email protected] b Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, P. R. China † Electronic supplementary information (ESI) available: Experimental details and additional figures. See DOI: 10.1039/c4cc04533e
13066 | Chem. Commun., 2014, 50, 13066--13069
Scheme 1
Construction of CPT@GO-CD–HA-ADA supramolecular assembly.
CD, a class of cyclic oligosaccharides, and hyaluronic acid, a widely distributed glycosaminoglycan, are all biocompatible, while the pristine GO-CD could be disrupted from several hundreds to tens of nanometres, and the resultant uniform and small-sized GO nanosheets may facilitate its biodegradation in the intracellular and extracellular environments.7 Secondly, the targeting ability derives from the hyaluronated adamantane (HA-ADA) chains, in which the HA skeleton can specifically recognize the HA receptorexpressing tumor cells in cancer metastasis (Fig. S1 in the ESI†).8 Thirdly, anticancer drugs, such as camptothecin (CPT), could be readily integrated into the water-soluble nanocarrier of GO-CD–HAADA (Scheme S1, ESI†). Consequently, this highly stable and compatible nanostructure is considered as an effective therapeutic for oncological treatment. The chemical structure and synthetic route for the preparation of the CPT@GO-CD–HA-ADA conjugate are shown in Scheme 1. For a biocompatible purpose, the size of GO sheets was controlled around 300–500 nm by simply adjusting the oxidation conditions
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and the reaction time during the chemical exfoliation process.9 Subsequently, the GO nanosheets were functionalized by grafting b-CD units via an amine-epoxy reaction.10 Moreover, benefiting from a two-step orthogonal noncovalent interaction, i.e., the supramolecular complexation of b-CD cavity with the adamantyl group and the p–p stacking interaction between the planar GO surface and the aromatic ring of the drug molecule, CPT, a cytotoxic quinoline alkaloid in cancer chemotherapy, was successfully endowed with water solubility and the targeting ability to bind malignant cells. The qualitative characterization for the formation of GO-CD–HAADA complex began with UV/vis, FTIR, and TGA experiments. In comparison to free GO, the absorption of GO-CD resulted in an obvious bathochromic shift from 227 to 256 nm, indicating the recovery of coplanarity to some extent after the removal of epoxy groups on GO sheets.11 In addition, no spectral change was observed upon complexation with HA-ADA, implying that the introduction of optically transparent HA-ADA component cannot affect the integrity of the GO-CD platform (Fig. S2 in the ESI†). Accordingly, some characteristic vibrations at 2928, 1155, 1078, and 1653 cm 1 in the FTIR spectrum of GO-CD–HA-ADA, corresponding to C–H and C–O in CD units and CO–NH in the HA-ADA polymer, respectively, jointly confirm the noncovalent modification of graphene sheets with the HA polymer by inclusion complexation (Fig. S3 in the ESI†). Furthermore, as can be seen from Fig. S4 (ESI†), in spite of a similar change profile, the decomposition temperatures of GO-CD–HA-ADA always situate themselves between the ones of GO-CD and HA-ADA in the region from room temperature to 800 1C, suggesting that the binary complex maintained its thermal stability compared to its precursors. The morphological information of the supramolecular assembly was visually obtained using atomic force microscopy (AFM) and dynamic light scattering (DLS) measurements. As can be seen from Fig. 1a, free GO gave a monodisperse nanosheet with an average height of 1.1 nm, corresponding to the completely exfoliated GO single layer. In addition, the height of GO-CD conjugate increased to 2.5 nm, which was in accordance with the attachment of CD units (height 0.8 nm) onto both sides of the GO surface (Fig. 1b). Moreover, an average height of 4.5 nm with a lateral dimension ranging from 50 to 150 nm in the AFM image of the GO-CD–HA-ADA complex was attributed to the cross-linking of GO-CD with the HA-ADA chain through intermolecular communication (Fig. 1c). In contrast, the cross-section analyses reveal that after loading CPT, the height of the ternary assembly of CPT@GO-CD–HA-ADA was ca. 6.4 nm, larger than that of GO, GO-CD, and GO-CD–HAADA, which would be originated from the spatial arrangement of CPT with the monolayer GO-CD and b-CD-appended HA polymer (Fig. 1d). Along with the microscopic investigation results, it is also found that there was an obvious morphological change in aqueous solution; that is, the average hydrodynamic diameter of GO-CD was 400 nm, whereas the one of GO-CD–HA-ADA was decreased to 80 nm at the same concentration, further suggesting that the continuous ultrasonication in the presence of the HA-ADA polymer could crack the GO sheet into small-sized components and then promote the eventual receptor-mediated internalization of the biocompatible supramolecular complex by cells (Fig. S5 in the ESI†).
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Fig. 1 Typical AFM images of (a) GO, (b) GO-CD, (c) GO-CD–HA-ADA, and (d) CPT@GO-CD–HA-ADA, respectively.
Subsequently, quantitative evidence regarding the composition and structure of CPT@GO-CD–HA-ADA was further provided by X-ray photoelectron spectroscopy (XPS). As expected, all the peaks of associated elements were recorded in the XPS survey spectra, where Na+ was introduced as the counterion of carboxylate groups on GO and HA backbones (Fig. 2 and Fig. S6–S7 in ESI†). Taking the CPT@GO-CD–HA-ADA conjugate as an example, the binding energy positions at 285, 398, and 530 eV were assigned to C1s, N1s, and O1s, respectively (Fig. 2c). The detailed XPS analyses are described in
Fig. 2 XPS survey spectra of (a) GO-CD, (b) GO-CD–HA-ADA, and (c) CPT@GO-CD–HA-ADA, respectively.
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the ESI.† Moreover, through a calculation based on the content ratio of nitrogen atoms in GO-CD (0.67%), GO-CD–HA-ADA (2.13%), and CPT@GO-CD–HA-ADA (2.36%), it can be verified that the mass fractions of CD in GO-CD and HA-ADA in GO-CD–HA-ADA were 27.7% and 40.5%, while that of CPT in CPT@GO-CD–HA-ADA was 3.7%, respectively. Therefore, the loading ratio of CPT on the GO surface was obtained as 3.7%, which was in good agreement with the UV/vis spectroscopic titration results as described below. It is significant to note that GO-CD–HA-ADA exhibited an excellent stability in both saline and serum environments, and this biocompatible property can be readily distinguished by the change of solution state after centrifugation. As shown in Fig. S8 (ESI†), GO-CD in phosphate buffer solution (PBS) and bovine serum albumin (BSA) was much less resistant to centrifugal force, as compared to the resultant complex that was stably dispersed under the same experimental conditions. This large disparity in the stability of GO-CD and GO-CD–HA-ADA implies that the formation of inclusion complex can greatly deter the negatively charged GO planes from the undesirable self-associated aggregation in aqueous solution. Consequently, in the CPT loading test, it is found that the solution of CPT@GO-CD could cause a very serious precipitation in PBS; while the one in the presence of HA-ADA still remained homogeneous and clear even after standing for 10 h (Fig. S9 in the ESI†). As a consequence of the favourable p-stacking interaction, CPT can be conveniently introduced onto the GO surface. As expected, a new band at 366 nm was observed in the UV/vis spectrum of CPT@GO-CD–HA-ADA, which is the characteristic absorption of CPT (Fig. 3).12 Meanwhile, the emission intensity of CPT was decreased by 92% after loading onto GO, mainly due to the high fluorescence quenching efficiency of GO sheets (Fig. S10 in the ESI†).13 In our case, the absorption of GO-CD–HA-ADA as the reference could be actually deducted from the UV/vis spectrum of CPT@GO-CD–HA-ADA, and then the net concentrations of CPT and GO-CD–HA-ADA were accordingly calculated using their molar absorption coefficients at 366 and 800 nm, respectively (see the ESI† for details). Therefore, the drug loading efficiency was obtained as
Fig. 3 UV/vis absorption of GO-CD–HA-ADA and CPT@GO-CD–HAADA, respectively ([GO-CD–HA-ADA] = 64.0 mg mL 1). Inset: release profile of CPT from the CPT@GO-CD–HA-ADA conjugate in vitro in PBS (pH 7.2, I = 0.01 M) at 37 1C.
13068 | Chem. Commun., 2014, 50, 13066--13069
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3.3%, which was quite similar to the XPS analysis results (3.7%). Moreover, the releasing behaviours of CPT from the GO-CD–HA-ADA nanoplatform were examined using fluorescence spectroscopy in PBS at 37 1C, providing a controlled and sustained release of CPT over a period of 10 h (Fig. 3, inset). Comparatively, the rate of drug release was much faster at pH 5.7, and this pH-responsive releasing behaviour could definitely inhibit the growth and reproduction of tumor cells in cancer cell environments (Fig. S11 in the ESI†). To further explore the molecular binding mode of CPT with GO-CD and HA-ADA, some control experiments were performed. As illustrated in Fig. S12 (ESI†), when CPT was mixed with GO-CD, the absorption peak at 366 nm was reproduced, which resembled the one in the CPT@GO-CD–HA-ADA system. Instead, no absorption beyond 300 nm was observed in the case of CPT with HA-ADA, convincingly proving that p-stacking interaction is the primary driving force to trap drug molecules onto the GO surface and the existence of HAADA cannot make any negative impact on the loading ratio of CPT (Fig. S13 in the ESI†). Finally, cytotoxicity experiments were carried out to evaluate the antitumor activity of the ternary assembly in vitro. Gratifyingly, superior to CPT and CPT@GO-CD, CPT@GO-CD–HA-ADA displayed a better anticancer activity toward MDA-MB-231 cancer cells, a type of human breast cancer cells with abundant HA receptors being overexpressed on its surface (Fig. 4a and Fig. S14a in the ESI†).14 After a 48 h incubation, the relative cellular viability of MDA-MB-231 for CPT@GO-CD–HA-ADA was 51.5%, which was lower than the corresponding value for free CPT (64.5%). Significantly, the relative
Fig. 4 Relative cellular viability and cell photos of (a–g) MDA-MB-231 and (h–m) NIH3T3 cell lines after the treatment with blank (b and i), CPT (c and j), GO-CD–HA-ADA (d and k), CPT@GO-CD–HA-ADA (e and l), CPT@GO-CD– HA-ADA with an excess of HA (f), and CPT@GO-CD (g and m), respectively, in 48 h incubation ([CPT] = 1.0 mM). The statistically significant differences were indicated with asterisks (P o 0.05).
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cellular viability of normal fibroblasts NIH3T3 with CPT@GO-CD– HA-ADA was 82.5%, which was quite high compared to that with free CPT (63.0%). Furthermore, when the receptors on the cancer cell surface were blocked by an excess amount of HA, the nanocarrier lost its original cell selectivity, giving a cellular viability that was nearly the same as the value obtained using free drugs. In particular, it is a remarkable fact that neither abnormal apoptosis nor cellular morphological change was observed in the GO-CD–HA-ADA group (Fig. 4d and k). These results jointly demonstrate that the obtained GO-CD–HA-ADA carrier could be utilized as a safe and promising candidate for drug delivery and other biomedical research. In conclusion, a tumor-targeted delivery system for CPT was constructed by the influence of supramolecular positive cooperativity of GO-CD with HA-ADA, displaying a satisfactory stability and biocompatibility under physiological conditions. The CD–ADA inclusion complex can largely prevent the GO skeletons from intermolecular aggregation in water, which then facilitate the disruption of a pristine GO sheet into small-sized components under ultrasonic conditions. Moreover, as investigated using cytotoxicity experiments, it can be seen that the ternary assembly of CPT@GO-CD–HA-ADA exhibited a higher curative effect and a lower cytotoxicity than a free drug. In principle, the simple loading and specific delivery of CPT in this work further stress the importance of facile synergetic interaction in the design and engineering of macrocycle-based drug carriers, by which the conventional chemotherapeutic drugs can be specifically delivered to their intended sites of action and their general toxicity can be reduced to a significant extent for wider clinical application. We thank the 973 Program (2011CB932502) and NNSFC (91227107 and 21102075) for financial support.
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