Guilong
Zhang‡
a,
Ruohong
Du‡
b,
Junchao
Qian
c,
Xiaojia
Zheng
b,
Xiaohe
Tian
d,
Dongqing
Cai
a,
Jiacai
He
b,
Yiqun
Wu
e,
Wei
Huang
e,
Yuanyin
Wang
b,
Xin
Zhang
f,
Kai
Zhong
c,
Duohong
Zou
*be and
Zhengyan
Wu
*a
aKey Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, People's Republic of China. E-mail: zywu@ipp.ac.cn
bDepartment of Dental Implant Center, Stomatologic Hospital & College, Key Laboratory of Oral Diseases Research of Anhui Province, Anhui Medical University, Hefei 230032, People's Republic of China. E-mail: zdhyy@ahmu.edu.cn
cHigh Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, People's Republic of China
dSchool of Life Sciences, Anhui University, Hefei 230601, People's Republic of China
eThe Department of Oral Surgery, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, National Clinical Research Center of Stomatology, Shanghai 200001, People's Republic of China
fSchool of Life Sciences, Anhui Agricultural University, Hefei 230036, People's Republic of China
First published on 27th November 2017
Considering the chemical exchange between gadolinium centers and water protons, nanosystems comprising gadolinium conjugated with high specific area nanocarriers might serve as more robust clinical tools for diagnosis and imaging-guided therapy. Herein, a pH-responsive nanosystem containing graphene oxide conjugated with a folic acid- and gadolinium-labeled dendrimer (FA-GCGLD) to boost its T1 contrast ability was developed, and doxorubicin (DOX) and colchicine (COLC) were efficiently loaded onto this nanosystem (FA-GCGLD-DOX/COLC). This nanosystem showed a prominent T1 contrast with an ultrahigh relaxivity of up to 11.6 mM−1 s−1 and pH-responsive drug release behavior. HepG2 cells treated with FA-GCGLD-DOX/COLC were efficiently inhibited, and the cell contrast was enhanced. In vivo, the tumor accumulation of FA-GCGLD-DOX/COLC significantly increased, thereby facilitating the systemic delivery of particles and exerting tumor growth inhibition and an enhanced tumor contrast effect. Moreover, compared to free drugs, FA-GCGLD-DOX/COLC effectively decreased the drug resistance of the tumor, thereby improving the cancer chemotherapeutic efficacy. In addition, injecting rats with FA-GCGLD afforded excellent magnetic resonance angiography (MRA) images with high-resolution vascular structures because of the long blood circulation time of FA-GCGLD. Thus, this study provides a powerful tool for diverse applications in the biomedical field, including accurate diagnosis and chemotherapy of tumors and the detection of cardiovascular diseases.
To compensate for these shortcomings, nanocarriers that can load Gd chelates have been developed.10,11 These nanocarriers could significantly enhance the Gd payload and increase the T1 relaxivity per nanoparticle. A wide range of nanocarrier-labeled Gd chelates have already been tested and have shown high contrast ability, including dendrimers,12–15 polymers,16–18 silica nanoparticles,19 and carbonaceous materials.20–22 Arguably, any significant future improvements in the r1 per particle will be achieved by developing a nanocarrier that supports larger Gd payloads. Graphene oxide (GO) possesses a high specific area and diverse functional groups and therefore could be used to enhance the Gd payload by conjugating gadolinium chelates in the intraparticle space. In addition, based on the Solomon–Bloembergen–Morgen (SBM) theory, the strategies for enhancing the relaxivity per Gd mainly have optimized the water residence time, increased the number of bound water molecules, and prolonged the rotational correlation time. Therefore, GO could also affix plenty of water molecules on its surface and optimize the contact efficiency between water protons and gadolinium chelates, thereby achieving an enhanced MRI contrast ability. However, according to the reported studies,23–25 the Gd payload was still low because of the direct grafting between gadolinium chelates and GO. Moreover, these nanosystems were comparatively larger and thus were hardly applied in vivo. Therefore, there is an urgent need to develop GO-based CAs with a high Gd payload and suitable size.
Conversely, GO-based drug delivery systems (DDSs) have attracted widespread attention and displayed an excellent cancer therapeutic effect in animal models.26–29 However, such systems suffer from several issues, such as low permeability,30 retention effect,31 and ligand recognition,32 which have seriously limited their further clinical use. One approach to resolve these problems is to develop DDSs with active targeting ligands and controlled-release ability. Mimicking the responsiveness of a living organism has yielded GO-based DDSs that sensitively respond to the microenvironment of the cancer cells of organisms; currently, these DDSs appear to be feasible strategies for enhancing the drug utilization efficiency and reducing the side effects. However, this approach might contribute to the problem of drug resistance, causing medicines to lose their potency against tumor tissue. Therefore, more efficient delivery strategies are needed.
In this study, we demonstrate a highly efficient theranostic system that was obtained by tailoring GO (TGO) conjugated with a folic acid- and gadolinium-labeled dendrimer (FA-GCGLD). Two types of anticancer drugs (doxorubicin, DOX and colchicine, COLC) were effectively loaded onto FA-GCGLD via π–π interactions with a loading capacity of up to 154%. Meanwhile, the release of these anticancer drugs could be triggered under acidic conditions, and the release rate increased as the pH decreased. Compared with Gd-DTPA, the T1 relaxivity of FA-GCGLD was greatly enhanced, which could be attributed to the high Gd payload and contact efficiency between the Gd centers and water protons. Cell assays indicated that this nanosystem enables not only the intracellular drug release but also the detection of the targeted accumulation of nanodrugs by MRI. The systemic delivery of drugs loaded in FA-GCGLD significantly enhanced the drug efficacy and inhibited the tumor growth. Moreover, the tumor contrast was dramatically enhanced, thereby improving the accuracy of liver cancer diagnosis. In addition, FA-GCGLD nanosheets with ultrahigh T1 relaxivity also showed a clear vascular structure by magnetic resonance angiography (MRA), indicating this nanosystem as a potential CA candidate in the future for the diagnosis of cardiovascular diseases.
![]() | ||
Fig. 1 Schematic diagram of (a) the preparation of FA-GCGLD-DOX/COLC and (b) its subsequent use for the accurate diagnosis and therapy of cancer in vivo. |
GO was fabricated from graphite powder via the classical Hummer's method.33 The original GO displayed a 2D sheet-like morphology (Fig. S1a†) and a lateral width of approximately 1.5 μm (Fig. S1c†), which was too large for in vivo applications. Subsequently, the original GO nanosheets were further tailored to afford smaller nanosheets of different shapes (Fig. S1b,†Fig. 2c). The analysis of the particle size distribution showed that the size of GO was reduced from 692 nm before tailoring to 108 nm after tailoring (Fig. S1d†). In addition, the atomic force microscopy (AFM) images show that the thickness of TGO was still less than 1 nm (Fig. 2a), indicating that TGO was a single layer and could be utilized in vivo. After FA-GLD was conjugated to the TGO nanosheets, energy-dispersive X-ray (EDX) analysis showed an obvious peak for the Gd element, implying that considerable FA-GLD was conjugated to the surface of TGO (Fig. 2d). Elemental mapping and surface scanning analysis demonstrated the uniform distribution of FA-GLD within the TGO nanosheets (Fig. 2b).
The hydrodynamic diameter of the particles was measured via dynamic light scattering (DLS). As shown in Fig. 3a, FA-GLD particles displayed a very small size of approximately 5.7 nm. TGO was 108 nm, and the size of the FA-GCGLD composite further increased to 186 nm, further indicating that abundant FA-GLD particles were successfully loaded onto the TGO nanosheets. In addition, Fig. 3b reveals that the size of FA-GCGLD did not increase over time and that no precipitation occurred, indicating that FA-GCGLD possessed good stability. Furthermore, we investigated the process of conjugating FA-GLD onto the GO nanosheets via zeta potential analysis (Fig. 3c). The FA-GLD particles had a mean charge of 12.5 mV, and TGO possessed a mean charge of −46.5 mV. After FA-GLD was conjugated to TGO, the charge of the particles became −8.7 mV.
In this context, the modification process of FA-GCGLD was investigated via ultraviolet visible (UV-Vis) and Fourier transform infrared (FT-IR) spectroscopy. As shown in Fig. 3d, GO possessed a typical absorption peak at 227 nm. Moreover, the absorption peak after the loading of GLD onto TGO shifted to 276 nm, indicating that the oxide groups in TGO were reduced or removed during the binding of the GLD particles. These findings suggested that the GLD particles were successfully loaded onto the TGO lattice. In addition, the new peak at 295 nm exhibited by FA-GCGLD suggested that FA-GCGLD featured many folic acid targeting ligands. For FA-GCGLD-DOX/COLC, the strong peaks at 355 and 496 nm were assigned to the absorption of COLC and DOX, respectively, which suggested that a considerable amount of drugs were loaded onto the FA-GCGLD nanosheet. In the FT-IR spectra (Fig. 3e), the FA-GLD peaks at 568, 1483, 1652, and 3078 cm−1 were ascribed to the Gd–O stretching vibration, benzene skeleton vibration, –CO stretching vibration, and
C–H stretching vibration, respectively, which indicated that GLD and folic acid were attached to the dendrimers. The TGO peak at 1728 cm−1 corresponded to the –COOH stretching vibration; this functionality was used to conjugate FA-GLD via an amide bond. For FA-GCGLD, the –COOH peak shifted from 1728 to 1649 cm−1, indicating the successful formation of the amide bond between FA-GLD and the TGO nanosheet. For FA-GCGLD-DOX/COLC, the new peaks appearing at 1256, 1086, 990, and 815 cm−1 suggested that many drug molecules were loaded onto the nanosystem. These data were consistent with the results from the UV-Vis analysis.
The surface composition, structure, and functional groups of the FA-GCGLD nanosheet were also investigated via X-ray photoelectron spectroscopy (XPS). A full spectral analysis confirmed the presence of C, N, Gd, and O elements in FA-GCGLD (Fig. 3f). The C 1s spectrum showed three peaks at 284.8, 286.7, and 288.2 eV (Fig. 3g), which were ascribed to graphite C (sp2 –CC–), C–O–C, and the amide bond, respectively. In addition, the N 1s spectrum possessed two peaks at 399.8 and 402.1 eV (Fig. 3h), which were assigned to the –C–N– bond in the dendrimer and the amide bond (HN–C
O), respectively. The strong peak that appeared at 1188.6 eV suggested that many Gd ions had been attached to the FA-GCGLD nanosheet (Fig. 3i). Therefore, these data suggested that the FA-GLD particles had been successfully grafted onto GO nanosheets via amide bond formation.
To achieve high MR contrast enhancement, assessing the gadolinium content in the FA-GCGLD nanosystem was necessary. The gadolinium content of FA-GCGLD was found to depend on the ratios of the dendrimer to TGO, with the highest gadolinium payload of approximately 34.2 wt% being achieved when the mass ratio of the dendrimer to TGO was 10/1 (Fig. 4a). In addition, based on the FT-IR spectrum (Fig. S3†),37 each TGO nanosheet was calculated to have 102 carboxyl groups, indicating that each nanosheet could load 102 FA-GLD particles. Thus, approximately 3264 Gd ions were theoretically attached to each TGO nanosheet. In actuality, the number of Gd ions on a TGO nanosheet was 2302, as determined by inductively coupled plasma optical emission spectrometry (ICP-OES).
We then studied the contrast enhancement ability of the nanosheets using MR tubes with different concentrations of gadolinium, which provided intuitional views of the contrast ability by distinguishing the brightness (T1) of the images. Three samples (Gd-DTPA, FA-GLD, and FA-GCGLD) with different concentrations were prepared and used in a T1-weighted MRI study (Fig. S7b†). In the T1-weighted imaging, both Gd-DTPA and FA-GLD showed a weak contrast (slight brightness), whereas FA-GCGLD at the same concentration exhibited a clear T1 contrast (strong brightness), indicating that FA-GCGLD had the best T1 contrast ability. The corresponding longitudinal relaxivity (r1) result of FA-GCGLD was 11.6 mM−1 s−1, which was 5-fold and 2-fold higher than that of Gd-DTPA (2.45 mM−1 s−1) and FA-GLD (5.48 mM−1 s−1, Fig. 4b). The high r1 of FA-GCGLD might be because of the fact that TGO possessed a high specific surface area and plenty of active groups could accelerate the natural affinitive binding to oxygen atoms in water molecules bonded to Gd species (Fig. S4†), thus resulting in an increased number of water molecules distributed on the TGO surface. To confirm our hypothesis, the T1 relaxation time of pure GO solution was measured (Fig. 4c). The results revealed that the T1 value of the solution gradually decreased as the GO concentration increased, indicating that GO could successfully affix water molecules and shorten the spin–spin time of the protons. Based on the average number of gadolinium atoms per FA-GCGLD nanosheet and the relaxivity per gadolinium atom, the relaxivity per FA-GCGLD nanosheet was approximately 2.7 × 104 mM−1 s−1. In addition, compared to Gd-DTPA and FA-GLD, the T1 relaxivity of FA-GCGLD was significantly enhanced.
The toxicity of imaging agents is also a key consideration for biomedical applications. Therefore, HepG2 and HeLa cells were used to evaluate the in vitro cytotoxicity of FA-GCGLD through the CCK-8 assay (Fig. S6†). When the cells were treated with FA-GCGLD in a wide range of concentrations for 24 and 48 h, the viability did not clearly decrease, indicating that FA-GCGLD had no significant cytotoxicity and showed good biocompatibility. The MR contrast enhancement of HepG2 cells administered with FA-FCGLD was then investigated. HepG2 cells treated with Gd-DTPA only slightly brightened as the concentration increased, with GCGLD showing similar results. However, FA-GCGLD caused a significant brightening of the cellular MR images, implying that FA-GCGLD could be effectively internalized by the HepG2 cells because of its folate-receptor targeting ability (Fig. S7a†). Furthermore, the reduction in T1 relaxation times observed from the relaxation time maps corroborated the T1-weighted image enhancements of HepG2 cells (Fig. S7c†). The HepG2 cells incubated with FA-GCGLD exhibited a significant decrease in the T1 relaxation time. Nevertheless, the cells incubated with Gd-DTPA and GCGLD particles showed only a slight decrease in the transverse relaxation time, further confirming that FA-GCGLD possessed an excellent targeting ability for HepG2 cells.
Next, the viability of the HepG2 cells incubated with free DOX/COLC, GCGLD-DOX/COLC, and FA-GCGLD-DOX/COLC was measured to evaluate the applicability of the nanosystem for cancer treatment. All groups were found to exhibit a dose-dependent cytotoxic effect. However, compared with GCGLD-DOX/COLC, the HepG2 cells treated with FA-GCGLD-DOX/COLC showed higher apoptosis at 24 and 48 h (Fig. 4f and S8†). This result might be due to the effective internalization of FA-GCGLD-DOX/COLC by the HepG2 cells arising from the folate-receptor targeting interaction, facilitating the release of drug molecules and thus inducing cancer cell death.
Subsequently, the precise subcellular localization and the dynamic entry of the FA-GCGLD nanosheets were evaluated via CLSM and transmission electron microscopy (TEM). The images obtained at 1 hour clearly showed that most of the FA-GCGLD nanosheets were closely associated with the cell plasma membrane and that negligible signal was observed from the intracellular region (Fig. 5b). With an increasing incubation time, FA-GCGLD crossed the cell membrane and penetrated into the cytosolic space. Notably, some FA-GCGLD nanosheets also permeated into the nucleus. Moreover, the images at 12 hours showed slightly less fluorescence remaining within the cells, implying a vesicular recycling process (e.g., exocytosis). In a parallel experiment, the TEM micrographs clearly exhibited the intracellular distribution of FA-GCGLD (Fig. 5d). Compared to an incubation time of 1 hour, the TEM images at an incubation time of 6 hours showed many nanoparticles in the cell. This result suggested that the cellular uptake of FA-GCGLD was time-dependent. In addition, the magnified images displayed abundant extracellular and intracellular vesicles that were closely associated with the plasma membrane (Fig. 5dc) and lysosomal/endosomal structures (Fig. 5dd), suggesting that the uptake mechanism of the FA-GCGLD nanosheets was endocytosis.
The inhibitory efficacy of the nanodrugs against tumors was evaluated in BALB/c nude mice (Fig. 6d). Mice injected with saline (control) showed rapid growth in their tumor size, suggesting that saline had no inhibitory effect on tumors. By contrast, DOX/COLC and GCGLD-DOX/COLC showed excellent inhibitory effects on tumors up to 34 days; however, at 34 days post-injection (p.i.), the tumor volume increased through explosive growth. This might be because the cancer cells generated gene mutations and thus possessed high drug resistance. Interestingly, the tumor volume of mice treated with FA-GCGLD-DOX/COLC was effectively inhibited and showed negligible growth throughout the whole treatment process, which could be attributed to the effective targeting ability of the particles and sustained drug release in the tumor tissue. These results indicated that the use of the targeted nanodrug prevented the emergence of tumor drug resistance and improved the cancer chemotherapeutic efficacy.
The results of the H&E staining of tumor biopsies treated with saline showed the presence of highly malignant hyperchromatic tumor cells on the slide (Fig. 7). However, the tumor cells on the slides treated with DOX/COLC, GCGLD-DOX/COLC, and FA-GCGLD-DOX/COLC showed different degrees of necrosis, and the malignant hyperchromatic area gradually decreased. Moreover, the extent of the necrotic areas was in the order of FA-GCGLD-DOX/COLC > GCGLD-DOX/COLC > DOX/COLC, whereas the size of the malignant hyperchromatic area was in the following sequence: FA-GCGLD-DOX/COLC < GCGLD-DOX/COLC < DOX/COLC. These results indicated that FA-GCGLD-DOX/COLC possessed the best therapeutic efficacy against tumor. In addition, the histological examination of the heart, kidney, liver, lung and spleen from mice treated with saline, GCGLD-DOX/COLC, or FA-GCGLD-DOX/COLC showed no or only minor histological changes, indicating that these samples were non-toxic or had minimal side effects on the body. In contrast, the samples treated with DOX/COLC presented obvious damage in the heart, kidney, liver, lung and spleen. Specifically, this damage consists of the following effects: (1) myocardial muscle cell lysis and inflammatory cell infiltration in the heart, (2) decrease in the glomerular interstitial space, (3) cloudy swelling of hepatocytes, (4) congestive change and mild thickening of the alveolar membrane, and (5) atrophy in the spleen. These results demonstrated that DOX/COLC could induce serious side effects on the body, whereas the FA-GCGLD nanosheet exhibited significantly enhanced therapeutic efficacy and reduced side effects.
![]() | ||
Fig. 7 Pathological analysis of various organs in mice injected with different samples via the tail vein. |
The ΔSNR values of FA-GCGLD within the hepatic portal vein and inferior vena cava at 15 min p.i. were 3.8-fold and 2.7-fold higher, respectively, than those of Gd-DTPA (Fig. 8d). Therefore, obtaining detailed diagnostic information from the vasculature using a single Gd-DTPA injection is difficult. FA-GCGLD could address many shortcomings of previous MRA CAs such as Gd-DTPA and achieve detailed vascular diagnostic imaging, which is particularly helpful in the context of a cardiovascular event. Therefore, the development of FA-GCGLD with a suitable size is likely to provide a new pool of molecules that might provide revolutionary solutions in the field of molecular imaging within a clinical setting.
Footnotes |
† Electronic supplementary information (ESI) available: Experimental details and supporting results. See DOI: 10.1039/c7nr07957e |
‡ These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2018 |