Article pubs.acs.org/Biomac
Carborane Derivative Conjugated with Gold Nanoclusters for Targeted Cancer Cell Imaging Jianling Wang,†,∥ Leifeng Chen,†,∥ Jing Ye,†,∥ Zhiyong Li,† Hui Jiang,† Hong Yan,*,‡ Marina Yu. Stogniy,§ Igor B. Sivaev,§ Vladimir I Bregadze,§ and Xuemei Wang*,†
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State Key Laboratory of Bioelectronics (Chien-Shiung Wu Lab), School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China ‡ State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China § A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov Street, Moscow 119991, Russia S Supporting Information *
ABSTRACT: Polyhedral borane derivatives have been utilized in the treatment of boron neuron capture therapy (BNCT) for brain glioma, and much attention has been paid to search excellent biocompatible boron-rich composites for effective cancer BNCT therapy. In this study, we have exploited the self-assembly of the gold nanoclusters with carborane amino derivatives (GNCs-CB) for the precise bioimaging of cancer cells and targeted delivery of this carborane compound to the tumors. Our observations demonstrate that the GNCs-CB can readily realize accurate tumor imaging and long-term accumulation in tumor sites by EPR effect and nanometer size effect, and thus efficiently implement tumor-targeting delivery of the carborane derivative and facilitate the real-time fluorescent visualization monitoring of the carborane targeted delivery process. This makes it possible to realize the accurate location of the tumor by the carborane derivative and reduce the damage to normal tissues in the process of boron neutron capture therapy through imaging guided treatment.
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INTRODUCTION Boron neutron capture therapy (BNCT) is known as an effective and powerful binary anticancer therapy in which boron compounds containing 10B isotope accumulate selectively in cancer cells and then are irradiated with thermal neutrons. The neutron capture reaction produces an excited 11B* nucleus, which undergoes a rapid fission reaction emitting the highenergy α-particle 4He2+ (1.47 MeV) and 7Li3+ ion (0.84 MeV) as well as low energy γ-rays (478 keV). The linear energy transfer (LET) of these heavily charged particles has a shortrange comparable with a cancer cell diameter. Therefore, the radiation damage can be limited only by the cancer cell minimizing the damage effects on the surrounding cells and tissues. Both targeted delivery of boron compounds into the tumor tissues and an accumulation at a level of 20−35 μg of 10B per gram of tissue are mandatory for efficient BNCT treatments.1−4 To achieve these requirements, hundreds of © 2017 American Chemical Society
low-molecular-weight boron cluster compounds (boranes and carboranes) were designed and synthesized.5−8 However, at present, only two compounds, disodium mercaptoundecahydro-closo-dodecaborate (BSH) and L-p-dihydroxyborylphenylalanine (BPA), are used in clinical trials. In this aspect, a promising strategy is the theory that nanomaterial-based drug delivery systems allow the accumulation of drugs into the tumor zone because of the so-called “enhanced permeability and retention” (EPR) effect.9,10 With this goal, carboranes have been conjugated to different nanomaterials including liposomes,11,12 dendrimers,13−15 nanoparticles,16−18 quantum dots,19,20 carbon nanotubes,21 and so on. Received: December 13, 2016 Revised: March 24, 2017 Published: March 28, 2017 1466
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CB in target tumor sites can facilitate accurate delivery of CB on tumor location and simultaneously bioimage the precise edge of the target tumor, thus reducing the possible damage to normal tissue in the process of BNCT and improving the therapy efficiency of BNCT treatment.
Fluorescent metal clusters including gold nanoclusters as a class of emerging fluorescent materials have attracted significant attention due to their unique physicochemical properties and subsequent strong photoluminescence. Our recent studies demonstrate that fluorescent gold nanoclusters (GNCs) or biosynthesized GNCs can efficiently accumulate in cancer cells and thus effectively facilitate tumor targeted imaging.22−28 The ability of BSH to form self-assembled monolayers on the gold flat surface29 and gold nanoclusters30 was reported almost 20 years ago. Meanwhile, the similar properties of mercapto derivatives of carboranes were also described in the literature.31−37 Herein we have exploited the self-assembly of the GNCs with the amino derivative of nido-carborane ([7NH2(CH2)3S-7,8-C2B9H11]−, CB, Figure 1). Our observations
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EXPERIMENTAL SECTION
Reagents. The carborane derivative (NH2NH3)+[7-NH2(CH2)3S7,8-C2B9H11]− was synthesized as described in the literature,44 stored at −18 °C and freshly prepared in aqueous solution immediately before use. Gold nanoclusters (GNCs) were freshly synthesized and dispersed in PBS (pH 7.2). Deuterited dimethyl sulfoxide and reduced glutathione were purchased from Sigma co., USA. Double-distilled water was used in all relevant experiments. Preparation and Characterization of Fluorescence Gold Nanoparticles (GNCs). Fluorescent GNCs were prepared according to the reported method using glutathione (GSH).45 Tetrachloroauric(III) acid (5 mL, 25 mmol/L) was added to freshly prepared GSH solution (5 mL, 25 mmol/L) at room temperature with constant stirring until the solution became colorless and transparent. After that, the stirring was stopped, and the reaction was kept away from light at room temperature for 2 weeks to obtain a pale yellow colloidal solution of gold nanoclusters. Afterward, the sample was centrifuged at 21 000g for 5 min to remove aggregates, and the supernatant was mixed with a certain amount of ethanol (Vwater/Vethanol, 2:1), which was centrifuged at 4000g for 5 min to collect the precipitate of GNCs. For characterization of GNCs morphology by TEM and fluorescence measurements, the GNCs were dispersed in ultrapure water. A transmission electron microscope (JEOL JEM-2100) was used to obtain TEM and HRTEM images, and a spectrofluorophotometer (RF-5301PC Shimadzu) was used to measure the fluorescence spectra. Preparation and Characterization of GNCs-CB Nanocomposites. A solution of CB (2.5 mmol/L, pH 7.2) was disposed by PBS. Then 1 mL of GNCs (15 mg/mL) and 1 mL solution of CB were added to 8 mL of PBS (pH 7.2) with thorough shaking for 15 min to obtain the nanocomposites of the gold nanoclusters−carborane derivatives. Meanwhile, in order to explore the influence of CB concentration on the fluorescence properties of the as-prepared GNCs, the CB solution is added drop by drop. The sample was airdried and subsequently examined by TEM, and the hydrodynamic diameter was monitored by dynamic light scattering (DLS) on a Zetasizer Nano ZS (Malvern Instruments). For NMR study, the CB (20 mg) was dissolved in DMSO-d6 (1.5 mL) and divided into 3 parts: A, B, and C. A was the control, while 10 μL of D2O and 10 μL of GNCs (15 mg/mL) in D2O were added to B and C, respectively. The 1H NMR spectra of A, B, and C were determined using a Bruker AM-500 NMR spectrometer.
Figure 1. Structure of the carborane derivative.
demonstrate that self-assembled GNCs-CB can efficiently implement tumor targeting delivery of carborane derivatives through the EPR effect and nanometer size effect and realize the real-time fluorescent visualization monitoring of the carborane-targeted delivery process by conjugated GNCs-CB (Scheme 1). Meanwhile, it is evident that the water-soluble CB can remarkably facilitate the fluorescence enhancement of GNCs, and the functional group −NH2 of CB can generate strong interaction with GNCs through self-assembly and thus form stable complexes of the GNCs-CB. The nanocomposites of the conjugated GNCs-CB showed relatively low toxicity and good cytocompatibility, while GNCs can effectively promote intracellular accumulation of CB and significantly increase the concentration of boron element in target cancer cells. In addition, nanocomposites of the self-assembly GNCs-CB could also be readily utilized for the in vivo fluorescent imaging of tumors, which have very good tumor targeting effect and can accumulate in tumor sites for a relatively long time (>48 h).38−43 This efficient accumulation of the fluorescent GNCs-
Scheme 1. Schematic Illustration of the Bioimaging Process for Cancer Cells by Using Self-Assembled GNCs-CB
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Figure 2. Characterization of GNCs. Left: Excitation and emission spectra of GNCs in aqueous solution, λexmax = 440 nm, λemmax = 585 nm. Inset: Pictures taken without and with excitation of a hand-held UV lamp (365 nm). Right: Typical TEM image of GNCs, showing the ∼2 Å interplanar spacing of the gold nanoclusters.
Figure 3. (A) The fluorescence emission spectra of GNCs (a) in the absence of CB and (b) in the presence of CB (300 μmol/L). (B) Plot of the GNCs fluorescence vs the concentration of CB in the mixture. (C) Panoramic TEM image of GNCs treated by CB; inset: photos in the absence or presence of under 365 nm illumination. (D) Enlarged TEM image illustrating the presence of gold nanoclusters, showing the ∼2 Å interplanar spacing of the gold nanoclusters. Cytotoxicity Assay of GNCs-CB. HeLa cells were seeded in 96well plates during logarithmic phase for MTT assay test. U87cells and L02 cells were treated similarly. After 24 h of incubation, different concentrations of GNCs, CB, and GNCs-CB were added to the culture plates, and further cultured for 48 h. The absorbance in 492 nm was measured by a microplate reader (MK3, ThermoFisher) for calculation of cell viability, according to the following equation:
Boron Distribution in Cells Treated with GNCs-CB. SEM-EDS (Zeiss, Ultra Plus) was used to explore the distribution of boron. HeLa cells in logarithmic phase were cultured in a 24 well-plate, which replaced the ITO glass (about 8000 cells/well) after 24 h culture, added to the GNCs-CB solution, and incubated for 24 h. Afterward, the HeLa cells were washed three times by PBS, then the remnant PBS was removed. 30% ethanol was slowly added to the HeLa cells, which was stored at 4 °C. After 15 min, the ethanol solution was removed. This process was repeated by using 50%, 70%, 80%, 90% and 100% ethanol solution, respectively. Finally, vacuum drying was done at 25 °C, and SEM was measured. Mice Model with Xenografted Tumor. Nude mice were originated from Health Science Center, Peking University, which were about 4 weeks old with a weight range of 18−22 g. The mice were bred in a super-clean lab for half a day in a dark and light cycle. They were injected with HeLa cells (∼5 × 106 cells) by subcutaneous tissue near their armpit. When the tumor grew to a size of 5−10 mm,
viability (%) = [Abs]test /[Abs]control × 100% where [Abs] means the absorbance at 492 nm. Cell Imaging. HeLa cells (∼105 cells/mL) of 1 mL were seeded in each well in culture plate for 24 h, then different concentrations of GNCs and CB were added successively. After incubation for 24 h, the cells were grown on a coverslip placed in the well. After removal of all culture media, the cells were fixed by 4% paraformaldehyde for 15 min. A Leica TCS SP5 confocal microscopy (Wetzlar, Germany) was used for cell imaging at an excitation wavelength 488 nm. 1468
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Biomacromolecules these mice with xenografted tumors could be used for further experiments. In Vivo Bioimaging Study. GNCs-CB solution of 0.1 mL was injected to mice model with xenografted tumor by tail. Then, after 24 h later, the fluorescence imaging was acquired on CRi Maestro by an excitation wavelength of 488 nm. The control group was examined under the same conditions.
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RESULTS AND DISCUSSION In this study, GSH was added as the reducing agent and stabilizer in the synthesis of GNCs. It is already known that
Figure 6. Laser confocal fluorescence images of HeLa cells treated with different species for 48 h: (A) control; (B) incubated with 25 nmol/L CB; (C) incubated with 150 ng/mL GNCs; (D) incubated with GNCs-CB containing 150 ng/mL GNCs and 25 nmol/L CB. Images were acquired at 400-fold magnification.
Figure 4. 1H NMR spectra of CB in the absence and presence of the GNCs, displaying the effect of the functional group −NH2 of CB treated by GNCs dispersed in D2O.
Figure 7. Upper panel: SEM images of HeLa cells: (A) incubation with 25 nmol/L CB; (B) incubation with GNCs-CB containing 150 ng/mL GNCs and 25 nmol/L CB. Lower panel: Table of the related elements content from EDS of a square region identified by the yellow or purple border in panels A and B.
Figure 5. HeLa cells viability after 48 h incubation with GNCs, CB, and GNCs-CB of various concentrations. Note that the concentration of GNCs remains 150 ng/mL in the relevant mixture during incubation with GNCs-CB.
Based on the above studies, we have further investigated the fluorescence spectra of GNCs upon addition of the CB. As shown in Figure 3A,B, it is noted that obvious fluorescence enhancement occurs when adding the CB to the GNCs solution, accompanying the apparent blue-shift of the relevant emission peak. The TEM characterization of the resulting GNCs-CB (Figure 3C,D) illustrates the good homogeneous dispersion of the related nanoclusters, whereas the HRTEM image showed the crystalline structure of the particles. It is evident that the self-assembly interaction of the GNCs with CB led to the formation of the conjugated GNCs-CB through the relatively strong interaction of the Au with the functional group −NH2 of CB (Figure 4), which can also readily result in obvious fluorescence enhancement (Figure 3). DLS analysis shows that the hydrodynamic diameter of GNCs-CB particles is ca. 9.4 ± 1.6 nm (Figure S1).
GNCs stabilized by glutathione have unique optical properties and excellent biocompatibility; especially, GNCs in vivo can be readily excreted out with liquid urine, which is a kind of metabolizable supersmall nanoparticle.41−47 Therefore, here we adopt GSH as the reducing agent and stabilizer for the synthesis of fluorescent GNCs, which is characterized by fluorescence spectroscopy and transmission electron microscopy (TEM), as shown in Figure 2. Our observations demonstrate that the as-prepared GNCs have good monodispersity (with particle size of ∼1.8 nm). HRTEM images demonstrate the crystallinity (with atomic lattice spacing of ∼2 Å) of the as-prepared GNCs. In addition, the GNCs have good fluorescence emission spectrum when excited at 440 nm (Figure 2), which is similar to those reported previously.48,49 1469
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Figure 8. Fluorescence imaging of HeLa xenograft tumor mice at various time points through tail-vein injection with GNCs-CB (0.1 mL) containing 0.15 mg/mL GNCs and 25 μmol/L CB in PBS. All images were obtained with the same instrumental parameters.
of two cell groups is good monolayer growth adhering to the wall, which further confirmed the good biocompatibility of CB and GNCs-CB, as also illustrated above in the cell toxicity assay. Especially, it is most important to notice from EDS analysis that boron content in cancer cells treated by CB is just 1.96% but that in target cancer cells treated by GNCs-CB is 6.98%, which indicates that GNCs can effectively promote boron accumulation in cancer cells, and thus lays the foundation for passive tumor targeting to effectively deliver boron contents in vivo for efficient BNCT treatment. Based on the cellular fluorescent imaging efficiency, we have explored the feasibility of bioimaging for targeted tumors in vivo by fluorescent GNCs-CB. We have established subcutaneously xenografted tumor mouse models to detect the possibility of in vivo fluorescence imaging of tumors through GNCs-CB. When the diameter of the tumor lump reached 7 mm, GNCs-CB was injected intravenously through the mice tails. After incubation for 24 h, the fluorescent signal was observed to cover the whole tumor, which can be precisely identified by its contour under the skin cover by fluorescence at 590 nm (Figure 8A). We gathered the picture at 28 and 48 h, and the fluorescence intensity in tumor was enhanced (Figure 8B and 8C), which indicated that GNCs-CB targeted accumulation in the tumor site by nanometer size effect and the EPR effect and bioimage for a relatively long time (>48 h).
For the exploration of the binding efficiency between the Au of GNCs with the functional group −NH2 of CB, 1H NMR spectroscopy characterization was further exploited in this study. As shown in Figure 4, the peak of the NH2 protons in CB is at ca. 4.5 ppm. Upon addition of the GNCs to the CB solution, the chemical shift of the relevant peak moves to ca. 4.0 ppm and becomes much sharper, while the chemical shifts of the CH groups in CB remain unchanged, which indicates the apparent interaction between the −NH2 group of CB and GNCs. In addition, considering the special nature of nanostructural surface of GNCs, there has already been many research reports in the literature about functional group interaction between GNCs and amino compounds (such as amino acid, protein, alkyl amine compounds, etc.),50−52 which agrees well with what we have observed for the self-assembed GNCs-CB. Furthermore, we have examined the cytotoxicity of GNCs, CB, and GNCs-CB against cancer cells using MTT assay to evaluate the biosecurity of the conjugated nanocomposites. As shown in Figure 5 and Figure S2, our MTT assay revealed that all of the relevant nanoclusters have good biocompatibility, even when treated at a concentration up to 15 μg/mL. HeLa and U87 cancer cells as well as L02 normal cells survived at different concentrations, with a viability of more than 85%. Low cytotoxicity and favorable biocompatibility of the conjugated GNCs-CB could provide a relatively safe source of boron reagents in BNCT treatment. The good biocompability and bright fluorescence properties of the GNCs-CB make it possible for further application in in vivo cancer cell bioimaging. As described above, based on the advantages of the conjugated GNCs-CB such as ultrasmall size, well-dispersed morphology, fluorescent characteristic, and low cytotoxicity, the delivery process of carborane derivatives could be readily monitored and traced by fluorescent GNCs-CB at the cellular level. As shown in Figure 6, it is evidenced that the GNCs and GNCs−CB were evenly distributed in HeLa cells after 48 h incubation; however, the cancer cells present very weak fluorescence in the control and CB, which can be ignored when compared with GNCs- or GNCs-CB-treated cells. In addition, SEM-EDS technique was also used to characterize the cancer cells treated by CB and GNCs-CB for 24 h, respectively. As shown in Figure 7, it is observed that the state
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CONCLUSION
In summary, in this contribution, good biocompatible and stable self-assembled GNCs-CB has been exploited for the precise bioimaging of cancer cells and target delivery of the carborane derivative to the tumors. Our observations demonstrate that the GNCs-CB can readily realize accurate tumor imaging and long-term accumulation in tumor sites by EPR effect and nanometer size effect. This provides a novel strategy for acquiring accurate location of the tumors by the carborane derivative and decreasing damage to normal tissues, which makes it possible to visually monitor boron source delivery to a tumor with fluorescent imaging simultaneously, and thus promoting the effect of BNCT treatment through imaging guided therapy. 1470
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b01845. Additional data for DLS histograms of nanoparticle size distributions, the MTT assay for the cell viability of U87 cells and L02 cells after 48 h incubated with GNCs, CB, and GNCs-CB of various concentrations (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hong Yan: 0000-0003-3993-0013 Xuemei Wang: 0000-0001-6882-7774 Author Contributions ∥
These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National High Technology Research & Development Program of China (2015AA020502), the National Natural Science Foundation of China (81325011, 21175020, 21327902, 21531004), and the collaboration grant of the Russian Foundation for Basic Research and the National Natural Science Foundation of China (16-53-53079 and 21611130027).
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ABBREVIATIONS CB, Carborane; GNCs, Gold Nanoclusters; GNCs-CB, Gold Nanoclusters with Carborane Derivatives; BNCT, Boron Neuron Capture Therapy; GSH, Glutathione; PBS, Phosphate Buffer Saline; NMR, Nuclear Magnetic Resonance; DMSO, Dimethyl Sulfoxide; DLS, Dynamic Light Scattering; SEM, Scanning Electron Microscope; EDS, Energy Dispersive Spectrometry; ITO, Indium Tin Oxide; ROI, Regions of Interest; TEM, Transmission Electron Microscopy; HRTEM, High Resolution Transmission Electron Microscopy; UV, Ultraviolet; MTT, Methylthiazolyldiphenyl-tetrazolium bromide; DMEM, Dulbecco’s Modified Eagle’s Medium; EPR, Enhanced Permeability and Retention
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REFERENCES
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DOI: 10.1021/acs.biomac.6b01845 Biomacromolecules 2017, 18, 1466−1472
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DOI: 10.1021/acs.biomac.6b01845 Biomacromolecules 2017, 18, 1466−1472
