Eu³⁺:Y₂O₃@CNTs—a rare earth filled carbon nanotube nanomaterial with low toxicity and good photoluminescence properties

Abstract

Red-emission phosphor europium-doped yttria (Eu³⁺:Y₂O₃) nanoparticles have been successfully filled into the nanocavity of carbon nanotubes (CNTs) via supercritical reaction and supercritical fluids followed by calcination. The existence of Eu³⁺:Y₂O₃ nanoparticles inside CNTs was characterized by TEM, EDS and XRD. The as-prepared nanomaterials (Eu³⁺:Y₂O₃@CNTs) exhibited strong red-emission at 610 nm, which corresponded to ⁵D₀ → ⁷F₂ transition within Eu³⁺ ions. It showed that the existence of the walls of the CNTs did not quench the luminescence of Eu³⁺:Y₂O₃. Due to the surface modification with Tween 80, Eu³⁺:Y₂O₃@CNTs had good water solubility. In vitro cytotoxicity studies showed that the as-prepared nanomaterials had low toxicity on HeLa cells at concentrations of 10–1000 μg mL⁻¹. And their use as luminescence probes for live cell imaging was demonstrated by using inverted fluorescence microscopy. With the advantages of the easy dispersion in water, low toxicity, and good photoluminescence (PL) properties, the as-prepared Eu³⁺:Y₂O₃@CNTs could potentially be used as nanophosphors in bio-imaging.

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Introduction

Benefited from their unique electronic, magnetic, and chemical properties arising from the 4f electrons,¹ rare-earth compounds have drawn great research attention in recent years.^2,3 Among the family of rare earth compounds, yttrium oxide (Y₂O₃) is a common used luminescent host material,⁴ and Eu³⁺:Y₂O₃ is considered to be one of the most promising red phosphors due to its excellent luminescence performance,^5,6 which has practical applications in high quality fluorescence lamps,⁷ emissive displays,⁸ in vivo biological imaging⁹ and so on. For the application of luminescent probes, the water-solubility and biocompatibility of nanoparticles are important.

Unfortunately, despite recent advances in synthetic methods for controlling the size and shape of Eu³⁺:Y₂O₃ nanoparticles,^5,10–20 many Eu³⁺:Y₂O₃ nanoparticles synthesized by a solution method are not directly used as biolabels due to their hydrophobic surface ligands, leading to their very low water-solubility. Additionally, the biological safety of the rare-earth nanomaterials remains controversial.^21–24 For instance, T. Andelman et al. demonstrated that the Y₂O₃ nanoparticles could cause cytotoxicity by increasing the amount of reactive oxygen species (ROS).²³ Sungho Lee et al. reported that the development of actin filament and cell proliferation could be inhibited by Y₂O₃ nanoparticles and the cytotoxicity of Y₂O₃ nanoparticles relied on their size.²² Therefore, it is necessary to develop a new and effective method to synthesize water-soluble or easily water-soluble and highly biocompatible Eu³⁺:Y₂O₃ nanoparticles for their application as biological fluorescent probes.

Carbon nanotubes (CNTs), which have hollow structures, can load functional materials inside the walls and combine the properties of CNTs with those of their guest components, to be used as delivery vehicles for drugs,^25–28 bio-imaging^29–31 and so on. The water-solubility of CNTs has been improved by sidewall covalent functionalization or noncovalent modification.^32,33 Moreover, in recent years, researchers have performed a lot of work on the cytotoxicity of nanotubes^34–49 and nanowires^50–57 and found that water-soluble functionalized carbon nanotubes had low toxicity.^48,49 Thus, CNTs can be used as an ideal carrier for rare-earth materials.

In this article, CNTs filled with Eu³⁺:Y₂O₃ nanoparticles were synthesized by a supercritical technique since this technique shows intriguing advantages for the synthesis of CNT-based composites, especially in filling materials into the nanocavity of CNTs.^58,59 The property and structure of the as-synthesized nanomaterials were characterized by TEM, EDS, XRD, and fluorescence spectroscopy. In order to demonstrate the potential application of the synthesized nanomaterials in bio-imaging, we have further functionalized the nanomaterials with Tween 80, to render them water-soluble. In vitro cytotoxicity studies of the synthesized Eu³⁺:Y₂O₃@CNTs were also undertaken.

Materials and methods

Materials

Y(III) nitrate hexahydrate (99.9%) and Eu(III) nitrate hexahydrate (99.9%) were purchased from Aldrich Chemical Co. The CCK-8 Cell Counting Kit and MTT cell proliferation and cytotoxicity assay kit were purchased from Beyotime Biological Co. Ltd. The Dulbecco’s Modified Eagle’s Medium (DMEM) cell culture medium and Fetal bovine serum (FBS) were purchased from Gibico Biologicals Inc. All of the other chemicals were of analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd. and were used without further purification.

Material synthesis

Functionalization of the carbon nanotubes. The CNTs used in this work were purchased from Chengdu Organic Chemicals Co. Ltd. The outer diameter was 10–20 nm, and their average length was 0.5–2 μm. The CNTs were oxidized and cut by refluxing in 60% HNO₃ for 12 h. After this reaction, the ends of the CNTs were opened and defects were formed on the side walls, resulting in carboxyl-containing CNTs.

Synthesis of Eu³⁺:Y₂O₃@CNT nanomaterials. Eu³⁺:Y₂O₃@CNTs were synthesized by a supercritical method, similar to the synthesis of Eu³⁺:Y₂O₃ nanoparticles,¹⁵ except that opened CNTs were added as the container for the rare-earth compounds. In a typical synthesis, 0.85 mmol of Y(NO₃)₃·6H₂O and 0.15 mmol of Eu(NO₃)₃·6H₂O were dissolved in 20 mL water and then mixed with 15 mL methanol. The mixed solution was put in a beaker under vigorous stirring for 10 min, and then the solution pH was adjusted to 7 by using 5 mL KOH solution. Finally, 40 mL of the above prepared mixture (volume ratio, water/methanol = 25/15) was transferred into a 100 mL stainless steel high-pressure reactor and mixed with 2 mg CNTs and heated at 400 °C for 10 min (the pressure was increased to 30 MPa). The filled samples were washed with distilled water for several times. The products were sonicated in 10 mL 65% HNO₃ solution several times to remove the rare-earth compounds attached to the surface of the CNTs and washed with distilled water and ethanol for three times and dried in air at 60 °C for 6 h. The final products were obtained through a heat treatment at 1000 °C in Ar for 2 h. For comparison, Eu³⁺:Y₂O₃ nanoparticles were also synthesized by a similar method.

Noncovalent modification of Eu³⁺:Y₂O₃@CNTs with Tween 80. In order to obtain better water solubility, Tween 80 was used to modify the as-synthesized nanomaterials. Noncovalent modification of Eu³⁺:Y₂O₃@CNTs was performed as follows: the as-prepared Eu³⁺:Y₂O₃@CNT nanomaterials were sonicated in an aqueous solution with 1% (v/v) Tween 80 for 0.5 h, followed by centrifugation to remove large aggregates and bundled nanotubes. The free Tween 80 was then thoroughly removed by repeatedly filtrating the mixture through 8–14 kDa filters (Millipore). The Tween 80-functionalized Eu³⁺:Y₂O₃@CNTs were finally re-suspended in phosphate-buffered saline (PBS). The as-prepared Eu³⁺:Y₂O₃ nanoparticles and pristine CNTs were also modified with Tween 80 using the same method.

In vitro cytotoxicity studies of the as-prepared nanomaterials in water. The in vitro cytotoxicity of the as-synthesized nanomaterials was evaluated using two methods: the Cell Counting Kit-8 (CCK-8, Dojindo) and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. In the CCK-8 assay, HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cells were seeded in 96-well plates (1 × 10⁴ cells per well). After 12 h of incubation, the medium was replaced with Tween 80-functionalized Eu³⁺:Y₂O₃@CNTs, Eu³⁺:Y₂O₃, and pristine CNTs, with different test concentrations (10, 100, 1000 μg mL⁻¹) in the culture medium. After these materials were added, the incubations were carried out at 37 °C and in 5% CO₂ atmosphere for another 12 h. Then the cell medium was removed and the dishes were washed by D-Hanks buffer solution for three times. One hundred microliters of CCK-8 solution were added to each well and incubated for an additional 2 h at 37 °C. The optical density (OD) of each well was recorded at 450 nm on a Microplate Reader (Infinite M200 Pro, A52769).

In the MTT assay, 20 μL MTT was added to each well after the cells were exposed to the nanomaterials and incubated at 37 °C for 4 h. The formazan product was then dissolved in DMSO and the absorbance of each well was recorded at 570 nm wavelength.

The cell viability (% of control) is expressed as the percentage of (ODtest − ODblank)/(ODcontrol − ODblank), where ODtest is the optical density of the cells exposed to the nanomaterials, ODcontrol is the optical density of the cells and ODblank is the optical density of the wells without HeLa cells.

Cellular imaging. HeLa cells (1 × 10⁴ cells) were seeded on glass coverslips that had been placed at the bottom of a culture dish. The HeLa cells were incubated for 12 h at 37 °C, and then the media was replaced by media that contained the Eu³⁺:Y₂O₃@CNTs (200 μL, 100 μg mL⁻¹). After 6 h incubation, the cells were washed three times with an excess amount of PBS and directly imaged using an inverted fluorescence microscope (AMG EVOS f1).

Characterization. The morphology of the samples was observed using a transmission electron microscope (TEM, Tecnai G2 F20 U-TWIN) operating on 200 kV. The crystal structure of the sample was determined by X-ray diffraction analysis (XRD, Bruker D8 Advance) using graphite-monochromized Cu Kα radiation (λ = 1.5406 Å). Energy dispersive X-ray spectroscopy (EDS) spectra were collected on Tecnai G2 F20 with an accelerating voltage of 200 kV. The contents of Eu and Y were determined by inductively coupled plasma mass spectrometry (ICP-MS) (PerkinElmer, Nexlon 300D). The photoluminescence spectra were measured by a spectra fluorophotometer using a 450 W xenon lamp (Fluorolog-3 FL3-21) at room temperature. Cellular imaging was obtained using an inverted fluorescence microscope (AMG EVOS f1).

Results and discussion

Fig. 1 shows the TEM and high-resolution TEM (HRTEM) images of the as-synthesized Eu³⁺:Y₂O₃@CNTs (Fig. 1a and b) and Eu³⁺:Y₂O₃ (Fig. 1c and d). It can be seen from the TEM image that the Eu³⁺:Y₂O₃ nanoparticles have been successfully inserted into the nanocavity of the CNTs via the supercritical synthesis process. The size and aggregation of the Eu³⁺:Y₂O₃ nanoparticles filled in the CNTs were confined by the nanocavity of the CNTs compared to the free Eu³⁺:Y₂O₃ prepared under the same conditions (Fig. 1b and c). The HRTEM image (inset of Fig. 1b) shows that there were no Eu³⁺:Y₂O₃ nanoparticles on the outside walls of the CNTs. The restricted inner nanocavity of the CNTs prevents encapsulated Eu³⁺:Y₂O₃ nanoparticles from forming a large scale crystal (Fig. 1d).

Fig. 1 TEM and HRTEM images of the as-synthesized Eu³⁺:Y₂O₃@CNTs (a and b) and Eu³⁺:Y₂O₃ (c and d).

The Eu³⁺:Y₂O₃ encapsulated CNTs were also analysed by energy dispersive X-ray spectroscopy (Fig. 2). The presence of the Y and O elements attributed to Eu³⁺:Y₂O₃. Since the Eu³⁺:Y₂O₃ nanoparticles attached to the outer walls of the CNTs have been carefully removed with acid, the existence of Y elements further confirmed that the Eu³⁺:Y₂O₃ had been filled into the cavity of the CNTs. The peak of the Eu element was not appearing due to the low content of Eu in the as-prepared nanomaterials. The accurate content of the Eu and Y elements was detected by ICP-MS (Eu 9 wt%, Y 42 wt%), and the atom ratio of Eu and Y was found out to be 1 : 8.

Fig. 2 EDS analysis of the as-synthesized Eu³⁺:Y₂O₃@CNTs.

In order to further investigate the crystal structure of the as-synthesized nanomaterials, XRD was employed. Fig. 3 shows the XRD patterns of the pristine CNTs, Eu³⁺:Y₂O₃@CNTs and Eu³⁺:Y₂O₃. The XRD diffraction peaks confirmed the crystallinity of the as-synthesized nanomaterials. The peaks located at 2θ values 25.7 and 43.2 can be assigned to the (002) and (100) diffractions of the CNTs, whereas the strong peaks located at 2θ values 20.4, 29.0, 33.7, 48.4 and 57.6 are considered to be the (211), (222), (400), (440) and (622) diffractions of cubic Y₂O₃ (JCPDS File 70-0603), respectively. The XRD pattern of the as-synthesized Eu³⁺:Y₂O₃@CNT nanomaterials exhibits both diffractions of the CNTs and Y₂O₃, and confirms the successful filling of Eu³⁺:Y₂O₃ nanoparticles into the nanocavity of the CNTs.

Fig. 3 XRD patterns of pristine CNTs, Eu³⁺:Y₂O₃, and Eu³⁺:Y₂O₃@CNTs.

The room temperature photoluminescence (PL) emission spectra of the as-prepared Eu³⁺:Y₂O₃@CNT nanomaterials and pristine CNTs are shown in Fig. 4. The strongest peak of the Eu³⁺:Y₂O₃@CNTs at 610 nm excited at 254 nm of UV-light corresponds to the forced electric dipole transition (⁵D₀ → ⁷F₂) within the Eu³⁺ ions, which is the typical spectral property reported for Eu³⁺:Y₂O₃ nanoparticles.²⁴ The peaks at 587, 592 and 599 nm are due to ⁵D₀ → ⁷F₁ transition, which are similar to those of Eu³⁺:Y₂O₃ nanoparticles synthesized using a supercritical method.¹⁵ The pristine CNTs show no peak between 570 nm and 670 nm under the same excitation conditions. Therefore, it can be concluded that the PL emission of the Eu³⁺:Y₂O₃ nanoparticles encapsulated in the hollow space of the CNTs is not quenched by the walls of the CNTs.

Fig. 4 Photoluminescence spectra of Eu³⁺:Y₂O₃@CNTs and pristine CNTs excited at 254 nm.

For the potential applications as probes for bio-imaging, the cytotoxicity of the as-synthesized nanomaterials was investigated. The cytotoxicity of Tween 80-functionalized Eu³⁺:Y₂O₃@CNTs was investigated in HeLa cells using CCK-8 and MTT assays. The viability of the untreated cells was chosen as the control. Fig. 5 shows the viability of HeLa cells treated by functionalized Eu³⁺:Y₂O₃@CNTs, Eu³⁺:Y₂O₃ and pristine CNTs at concentrations in the range of 10–1000 μg mL⁻¹. The results show a dose dependent decrease in their relative cell viability with the concentrations of the composite increased. The Eu³⁺:Y₂O₃@CNTs show low toxicity to HeLa cells even when the cells were exposed to a high concentration of 1000 μg mL⁻¹ for a period of 12 h while Eu³⁺:Y₂O₃ shows a rather higher cytotoxicity in the same concentration, which indicate that after encapsulated by CNTs the cytotoxicity of Eu³⁺:Y₂O₃ nanoparticles is reduced. The above results suggest that the as-synthesized Eu³⁺:Y₂O₃@CNTs can be used as potential probes for bio-imaging. The results also indicated that pristine CNTs are more toxic to HeLa cells than Eu³⁺:Y₂O₃ filled CNTs in the same weight concentration. According to the results of ICP-MS, the mass ratio of Eu and Y in Eu³⁺:Y₂O₃@CNTs was around 50%, and the number of CNTs in pristine CNTs was much more than in Eu³⁺:Y₂O₃@CNTs at the same weight.

Fig. 5 Cellular viabilities of HeLa cells exposed to functionalized Eu³⁺:Y₂O₃@CNTs, CNTs, and Eu³⁺:Y₂O₃ nanoparticles with different concentrations determined by CCK-8 assay (a) and MTT assay (b).

To demonstrate the potential of the as-prepared Eu³⁺:Y₂O₃@CNTs as probes for biological imaging, we incubated amphiphilic Tween 80-functionalized Eu³⁺:Y₂O₃@CNTs with HeLa cells. The fluorescence image (Fig. 6) shows that the Eu³⁺:Y₂O₃@CNTs exhibit good photoluminescence properties inside the cells.

Fig. 6 Overlay image of live HeLa cells after incubation with Eu³⁺:Y₂O₃@CNT nanomaterials (100 μg mL⁻¹) (red represents the emission from the Eu³⁺:Y₂O₃@CNTs).

Conclusions

In summary, Eu³⁺:Y₂O₃ filled CNT nanomaterials have been successfully synthesized by a supercritical technique, which is the first case that supercritical synthesis is used in the nanocavity of nanotubes. The as-synthesized Eu³⁺:Y₂O₃@CNTs were characterized by TEM, EDS, XRD and PL spectroscopy. With the excitation at 254 nm of UV-light, a special red-emission at 610 nm has been observed, which was from the Eu³⁺:Y₂O₃ nanoparticles encapsulated in the CNTs. Surface functionalization with Tween 80 enabled the synthesized materials to be dissolved in water and to have the possibility of further functionalization by other biomolecules. The Eu³⁺:Y₂O₃@CNT nanomaterials show low cytotoxicity in HeLa cells at concentrations of 10–1000 μg mL⁻¹. Using an inverted fluorescence microscope, we have demonstrated the application of Eu³⁺:Y₂O₃@CNTs as luminescent probes for live cells. With the properties of fluorescence emission, easily water-soluble and low cytotoxicity, the Eu³⁺:Y₂O₃@CNT nanomaterials have potential applications in bio-imaging. The supercritical synthesis method in filling the inner space of the CNTs could be applied to other rare-earth materials, such as up-conversion materials, which have greater potential in deep-tissue imaging.

Acknowledgements

The research was supported by National Basic Research Program of China (973 Program) (2012CB932601) and National Natural Science Foundation of China (21271174).

Notes and references

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