The isotopic effects of ¹³C-labeled large carbon cage (C₇₀) fullerenes and their formation process

Abstract

Fullerene, C₆₀, and its derivatives, have been fully investigated and commercialized. The importance of large carbon cage-based fullerenes for biomedical applications was gradually recognized due to their fantastic biological effects. In nanotoxicology, the key detection techniques are able to retain the intrinsic structure and properties of nanomaterials in a biological background. However, there were puzzling questions regarding how to facilely obtain detectable fullerene nanomaterials and their formation process. In this study, ¹³C-enriched fullerenes of a large carbon cage, C₇₀, were synthesized on a large scale from a ¹³C-enriched raw carbon material by an arc-discharge method. The stable isotope ¹³C was directly incorporated into the skeleton of the fullerene cages without destroying their intrinsic structures. The isotopic effects of ¹³C-labeled C₇₀ were investigated in detail. The amount of ¹³C labelling of C₇₀ was about 7% higher than that of the natural abundance, which greatly improved the ¹³C detection signal in isotope ratio mass spectrometry and gave an apparent 8-fold carbon nuclear magnetic resonance signal enhancement for the fullerenes. The ¹³C-enriched fullerenes showed significant isotopic effects, such as the strongest peak position shifting up (m/z > 840) and the Poisson distribution of the isotopic peaks in the mass spectra, and the migration or splitting of infrared and Raman characteristic peaks. When comparing the ¹³C labelling amounts and the isotopic effects of ¹³C-enriched C₇₀ and those of ¹³C-enriched C₆₀, the formation dynamics of the fullerenes were different with different carbon cages; lower carbon cage fullerenes were easily generated in the arc-discharge process, and the ¹³C stable isotopic effects in high carbon fullerenes were also slightly weak. Moreover, these important isotopic effects of the ¹³C-enriched fullerenes will facilitate the development of new analytical methods for carbon nanomaterials in vivo.

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Introduction

Fullerenes, one category of the most popular carbon nanomaterials, have been widely used in solar energy, cosmetics, personal care, electronics and biomedicine due to their unique chemical and physical properties.^1–7 Their biological efficiency depends on both the modification of the cage and the cage size (C₆₀, C₇₀, C₈₂, etc.). To date, pristine C₆₀ and its functionalized derivatives have been fully investigated and commercialized. The large carbon cage-based fullerenes possess much greater biological significance. For instance, C₇₀ and its derivatives exhibited higher photodynamic therapy and antioxidation efficiencies due to the much more extended conjugated π-system of the cage, which can efficiently absorb electrons.^8–12 Metallofullerenes of larger carbon cages (e.g. Gd@C₈₂ and Gd@C₈₂(OH)_x) also showed promise as radiotracers, magnetic resonance imaging (MRI) contrast reagents and anticancer drugs with high efficacy and low toxicity.^13–18 Obviously, these biomedical investigations of fullerene nanomaterials, especially for the large carbon cage-based fullerenes, are very significant.¹⁹ Inevitably, the related health and safety issues of fullerene nanomaterials have become urgent.^20–23 Nevertheless, the principal issue is how to quantitatively trace or detect intrinsic carbon nanomaterials in vivo.

Compared with C₆₀, the synthesis and chemical structure of the large carbon cage-based fullerenes have been less investigated, especially for the detectable labeled fullerene nanomaterials, including the large-scale synthesis of the labeled fullerenes and the influences of labeled groups, etc. Actually, it is very difficult to directly and quantitatively detect fullerene nanomaterials in vivo, because of the high carbon background in environmental and biological systems, the lack of specific detection signals of carbon nanomaterials and the complexity of biological matrixes.²⁴ Currently, radioactive labeling and fluorescent modification have been adopted to quantify fullerene nanomaterials in vivo.^25–32 Although these labeling methods have high sensitivity and specificity, radioactive labeling suffers from some inescapable drawbacks, such as the strict conditions of radioactivity operation and the generation of radioactive waste. Separately, fluorescent groups easily detach away from the modified nanomaterials or are quenched during exposure to environmental and biological systems. In addition, metallofullerenes, as higher temperature superconductors, redox active systems, and contrast agents etc., have been investigated.^{14,15,33–40} However, the properties and structures of fullerenes can be significantly altered by the metal inside the carbon cage.^33–40 The synthetic yield of metallofullerenes is also far less than fullerenes. Therefore, stable isotopic labeling is a good choice, which overcomes many of the drawbacks aforementioned.^41–50

Stable isotopic labeling combined with the high sensitivity and accuracy of isotope ratio mass spectrometry (IRMS) can provide information about the geographic, chemical and biological origins of substances, and has been widely used in diverse disciplines such as archaeology, medicine, geology, biology, food authenticity and forensic science for several decades.^{43–47,51–53} The ¹³C stable isotope can be incorporated into the skeletons of these carbon nanomaterials. ¹³C stable isotopes are used to investigate the structural properties and the formation mechanism of fullerene nanomaterials.^54–65 We have demonstrated previously the ¹³C stable isotopic labeling of carbon nanotubes (CNTs), carbon quantum dots and C₆₀ for in vivo quantitative biodistribution studies.^48–50 The ¹³C stable isotopes labeled directly on the skeleton of these carbon nanomaterials do not damage their stability and intrinsic structures. Therefore, the invaluable ¹³C stable isotopic tracers can reflect the real properties of carbon nanomaterials in biological systems and can also be potentially used as internal standard substances. Feasible ¹³C stable isotopic labeled technology will promote the development of carbon nanomaterials in nanotoxicology.

In the present work, taking C₇₀ as an example of a large carbon cage-based fullerene, ¹³C-labeled C₇₀ was synthesized on a large scale from a ¹³C-enriched raw carbon material by an arc-discharge method. The stable isotope ¹³C was directly substituted for the carbon atoms of the fullerene cages. The ¹³C-labeled C₇₀ samples were separated and purified through preparative high performance liquid chromatography (HPLC). The number of ¹³C atoms per C₇₀ cage was determined by IRMS. The isotopic effects of ¹³C-labeled fullerenes were studied in detail by mass spectrometry (MS), carbon nuclear magnetic resonance (CNMR), Fourier transform infrared (IR) and Raman spectroscopies, and an electrochemical system. The obtained ¹³C-enriched fullerenes, as tracers or potential internal standard substances, will be very important for the biosafety and biomedical development of fullerene nanomaterials in the future.

Experimental

Preparation of ¹³C-enriched fullerenes

¹³C-enriched fullerenes were prepared by an arc-discharge method.^{54,59,66–69} Briefly, the anode electrodes were fabricated by coring out natural-abundance carbon rods and packing them with isotope-enriched amorphous carbon powder (Cambridge Isotopes, 99% ¹³C). The cathode was a pure graphite rod. The arc discharge was performed under a He atmosphere (200 Torr) with the following parameters: interelectrode distance: 5 mm; current: 110 A; voltage: 27 V. Fullerene without labeling was prepared following the same protocol using normal carbon (98.5% ¹²C) as the starting material.

Separation and purification of ¹³C-enriched fullerenes

The carbon soot-containing fullerenes were collected and refluxed in CS₂ for 10 h to extract the ¹³C-enriched fullerenes. The ¹³C-enriched fullerenes were further separated and purified by high performance liquid chromatography (HPLC, LC908-C₆₀, Japan Analytical Industry Co.) with toluene as the mobile phase at a flow-rate of 15.0 mL min⁻¹ and employing a Buckyprep column (20 mm × 250 mm, Nacalai Co., Japan), equipped with a UV detector (wavelength, 335 nm).

Determination of ¹³C amounts in ¹³C-enriched fullerenes

IRMS can precisely measure the relative abundance of stable isotopes. Samples are converted into gas and then ionized. Subsequently, the ionized gases with different m/z values are separated under a magnetic field and quantified by the detectors. By comparing the detected isotopic ratios to an isotopic standard, an accurate determination of the isotope composition of the sample is obtained. Therefore, it can eliminate any bias or systematic error in the measurements. For example, carbon isotope ratios are measured relative to the internationally recognized C standard Vienna Pee Dee Belemnite (VPDB) and are reported in the delta notation, δ,
where the ¹³C/¹²C_standard ratio of the VPDB standard sample was 0.0112372. Aliquots of 0.2000 ± 0.0100 mg fullerene samples were weighed into Φ 3.2 × 4 mm tin cups for stable isotope analysis. Urea was the working standard, and the analyses of the carbon isotopes were carried out using a Finnigan MAT-253 (Thermo Electron-Finnigan, USA) continuous-flow isotope ratio mass spectrometer (ConFlo III, Finnigan MAT) coupled with a Flash Elemental Analyzer 1112 (Finnigan, Flash EA 1112 series).

Investigation of isotopic effects in ¹³C-enriched fullerenes

The detection of ¹³C incorporation is achieved by measuring the isotopic distribution and shift caused by the slightly heavier ¹³C atoms. To investigate the isotopic effects, the obtained fullerene samples were dissolved in toluene and characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS, Autoflex, Bruker Co., Germany) under negative ion mode, and microprobe Raman spectroscopy (Renishaw inVia plus, Renishaw, UK) under the conditions of 514 nm excitation wavelength, 0.5% laser power, 4 cycles and exposure for 50 seconds. The powdered fullerene samples were ground into KBr pellets under an appropriate pressure for Fourier transform infrared spectroscopy (IR, Tensor 27, Bruker, Germany). The fullerene samples for CNMR were dissolved in an excess of toluene-d₈ and evaporated at 25 °C until saturation was achieved. Using a 30 deg pulse and a 20 s pulse delay, a total of 30 720 accumulations obtained over 48 h using a Bruker AV 400 instrument (400 MHz, Switzerland) gave a spectrum with an acceptable signal/noise ratio, showing clearly the presence of fullerene. The analysis and comparison of all the data were normalized by a mathematical method using Origin software (OriginPro 8.0). For electrochemical measurements, 4.5 mg of C₇₀ was dissolved in 10 mL acetonitrile/toluene (volume ratio: 1 : 4) containing 0.1 M tetrabutylammonium perchlorate (TBAClO₄) electrolyte under 30 minutes of ultrasound. A CHI604E electrochemical workstation (Shanghai, China) was used for cyclic voltammetry (CV). A three-electrode cell was used with Ag/AgCl as the reference electrode, Pt wire as the counter electrode, and a glassy carbon electrode as the working electrode. The cyclic voltammetry measurements were recorded by a linear potential scan at a sweep rate of 50 mV s⁻¹. The samples were purged with high-purity nitrogen for 10 min before the test was performed; the potential range was from −2.0 to 0 V. All the electrochemical experiments were performed at 25 ± 1 °C.

Results and discussion

Separation and purification of ¹³C-enriched fullerenes

A Buckyprep preparative column can be used for the separation and enrichment of fullerene nanomaterials. CS₂ solvent extracts containing fullerenes were separated into C₆₀, C₇₀, C₇₆, C₈₂ and C₉₆ on the column in 100 minutes. There was a clear resolution of the C₆₀ and C₇₀ (R_s > 2) without interference from endogenous sources (Fig. 1). The retention times of C₆₀ and C₇₀ were 11.707 and 18.355 min, respectively. Herein, we did not observe the effect of stable isotope ¹³C labeling of the skeleton of the carbon cages on the chromatographic retention times of the fullerenes. ¹³C-enriched fullerenes and normal fullerenes have the same chromatographic behaviour. The yield of pure ¹³C-enriched C₇₀ was about 2–3% relative to the discharged soot.

Fig. 1 Separation and purification of ¹³C-enriched fullerenes.

MS analysis

The HPLC-isolated fullerenes were identified by MALDI-TOF-MS. The mass spectra for the purified fullerenes and ¹³C-enriched fullerenes are shown in Fig. 2. Normal C₇₀, due to the high abundance of ¹²C in nature, had a dominant peak at 840 (m/z), and was consistent with the theoretical calculation. C₇₀ with a high purity of greater than 99.5% was achieved. The ¹³C isotopes were labeled on the skeleton of the fullerenes. The MS spectrum of the ¹³C-enriched C₇₀ indicated clearly a statistical distribution of isotopic abundance. As shown in Fig. 2, compared to the C₇₀, the highest peak of ¹³C-enriched C₇₀ shifted due to ¹³C from 840.18 (m/z) to 845.75 (m/z). The isotopic distributions in the MS spectra were widened from 840 to 855 (m/z) and displayed the Poisson distribution extremely well. This experimental evidence was similar to that reported in the literature.^55,58,59 The MS of ¹³C-enriched C₆₀ was compliant with our previous reports.⁶⁹ In addition, we also found that other high carbon fullerenes (such as ¹³C-enriched C₇₆, C₈₂ and C₉₆) displayed similar results. The stable ¹³C isotope enriched the isotopic distribution in the mass spectra and the composition of the carbon elements of the fullerenes. The isotopic effect of ¹³C-enriched fullerenes clearly changed the isotopic distribution in the MS.

Fig. 2 MS spectra of ¹³C-C₇₀ (left) and C₇₀ (right).

IRMS analysis

Generally, stable isotope labeling is combined with IRMS, which is a specialization of mass spectrometry and can precisely measure the relative abundance of stable isotopes, then quantify ¹³C in fullerenes. ¹³C is an environmental and non-radioactive isotope of carbon in nature. It makes up about 1.1% of all the natural carbon on earth. IRMS can detect the ratio of ¹³C to ¹²C of the test samples, which is very sensitive for the addition of the ¹³C isotope into fullerene. Using IRMS we measured the δ of ¹³C-enriched C₇₀ from the different carbon isotope proportion of the synthetic materials (the mass ratio of ¹³C-powder/¹²C-rod was 15 : 85). The δ signal showed an increment of about 7000 with ¹³C/¹²C increasing by only 5 percent in abundance ratio (Table 1).

Table 1 ¹³C/¹²C isotope ratios of ¹³C-enriched C₇₀

Addition of ^13C in raw carbon powder (%)	δ ^13C/^12C	^13C/^12C (%)	^13C-enriched C70
0	−17.436	1.087	C70
15	7211.047	8.408	^13C5C65

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The number of ¹³C labels on the skeleton of fullerene was calculated and estimated by the following equation:

The value n_carbon is the total carbon number of the fullerene cage. ¹³C-enriched C₇₀ is expressed as ¹³C_nC_(70−n). The number of ¹³C atoms in a ¹³C-enriched C₇₀ molecule was 5, meaning ¹³C₅C₆₅. Compared to normal C₇₀, the abundance of ¹³C in ¹³C-labeled fullerenes was ∼7% higher than that of the natural abundance. The stable isotope ¹³C may be randomly distributed in the carbon cage.⁵⁶ The ¹³C labelling amount of ¹³C-enriched C₇₀ was ∼2% less than that of the ¹³C-enriched C₆₀ in our previous report.⁶⁹ The mixed carbon powders needed to be packed closely and uniformly. Generally, the extractions from the discharged carbon soot include many high carbon fullerenes (such as C₇₆, C₈₂ and C₉₆ etc.) except C₆₀. During the discharge process, the packed ¹³C powder was utilized dispersedly by the variety of fullerene carbon cages and other carbon substances (e.g. graphite, graphene, carbon nanoparticles). ¹³C was also labeled in other higher carbon fullerenes of the discharged carbon soot. So, the numbers of ¹³C atoms in C₆₀ or C₇₀ were less than the proportion of the starting materials. By comparison of the ¹³C labelling amounts in ¹³C-enriched C₇₀ and C₆₀, we found obviously that ¹³C was easily utilized by low carbon fullerene. We sought to understand the formation process of fullerenes by reasoning that low carbon fullerene was more easily generated, and the construction of the fullerenes probably originated from small carbon cages formed in the arc-discharge process. The speculations were consistent with the previous reports.^{55,56,59,61,64} The skeleton ¹³C-labeling technique has been facilitated to explore the formation mechanism of fullerenes and other carbon materials.^54–65 However, the skeleton ¹³C-labeled fullerenes were obtained largely from direct addition of the stable isotope in the origin by an arc-discharge method. The ¹³C labeling was stable without introducing other outside atoms onto the carbon networks. In the future, we believe that it is significant for exploring and optimizing the synthesis process to improve the ¹³C labelling amount in fullerenes.

NMR analysis

NMR spectroscopy is a powerful research technique for investigating the physical and chemical properties of chemical molecules (ranging from small compounds to large proteins or nucleic acids). The CNMR spectrum for ¹³C-enriched C₇₀, shown in Fig. 3, consists of five lines with intensities in the ratio 10 : 20 : 10 : 20 : 10, with chemical shifts of 130.5, 145.0, 147.0, 147.7, and 150.3 ppm, as reported by Johnson et al.^61,70 The spectrum strongly confirmed the D_5h C₇₀ structure with five chemically distinct kinds of carbon atoms, in good agreement with the spectrum of C₇₀. In addition, all the carbon atoms in the C₆₀ molecule were chemically equivalent; the CNMR spectrum for C₆₀ was a single line at 143 ppm (in both liquids and solids).^71–74 The single-resonance CNMR spectrum of C₆₀ was also strong evidence for the icosahedral symmetry of this molecule. In a magnetic field, the addition of ¹³C resulted in the NMR active nuclei enhancing the absorption of electromagnetic radiation at a frequency characteristic of the isotope. The sensitivity and signal strength of the CNMR spectra were all increased in the skeleton ¹³C-enriched fullerenes, while their chemical shifts were not changed in ¹³C-enriched C₆₀/C₇₀ (Fig. 3). The peaks corresponding to ¹³C-enriched C₇₀/C₆₀ and C₇₀/C₆₀ are all at the same positions, which is in excellent agreement with the results reported by other groups.^55,57,58

Fig. 3 NMR spectra of ¹³C-enriched fullerenes and unlabeled fullerenes.

From the results of the CNMR spectra, we did not observe that the symmetry of the molecular structure was altered or destroyed due to ∼7% ¹³C incorporation into the skeleton carbon cage of the fullerenes; they only show signal enhancement of the spectra and reduction of the data acquisition time to a feasible 3 days despite low solubility.^75,76 The signal strength of the CNMR of ¹³C-enriched C₇₀/C₆₀ indicated about an 8-fold increase with five ¹³C substitutions in the fullerene carbon cage. As expected, the proton NMR spectra of the samples dissolved in toluene-d₈ were devoid of any absorption besides the C₆D₅CD₃ peak at 137.11 ppm. In brief, these were the typical characteristics: that the introduction of ¹³C enlarged the CNMR signal of the fullerenes, and enhanced the detection sensitivity of the CNMR. The CNMR signal enhancement of the fullerene nanomaterials would provide the potential for the development of new analytical methods.

IR and Raman analysis

IR and Raman spectra are both molecular vibration spectra which reveal the characteristics of molecular structure. Compared to ¹²C, ¹³C has a relatively heavy atomic weight of 13, which is made up of 6 protons and 7 neutrons. The introduction of the heavy atom ¹³C would likely change the molecular vibrations of fullerene. The IR results revealed that the ¹³C-enriched C₇₀ retained all the characteristic bands of ¹²C₇₀ and showed a trend that with the increase of incorporated ¹³C atoms the absorption bands shifted slightly to lower wavenumbers (Table 2). Compared to C₆₀, the molecular symmetry of C₇₀ and ¹³C-enriched C₇₀ was lower. Their IR spectra were relatively complicated. The introduced ¹³C atoms did not cause strong changes of the IR spectra and made the most characteristic peak shift from 1427 cm⁻¹ to 1423 cm⁻¹ (Fig. 4).

Table 2 Displacement of the IR characteristic peaks of C₇₀ and ¹³C₅C₆₅

	C70	^13C5C65
νIR/cm^−1	1427.3	1423.4
	1132.2	1128.3
	792.7	792.7
	723.3	723.3
	671.2	669.3
	574.8	574.8
	532.3	532.3
	457.1	455.2

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Fig. 4 IR spectra of ¹³C₅C₆₅ and C₇₀.

For ¹³C-enriched C₆₀, the small addition of ¹³C atoms also did not change the four vibration modes, but made the characteristic bands shift down observably.⁶⁹

The Raman spectrum of ¹³C-enriched C₇₀ displayed the usual changes and is shown in Fig. 5. For C₇₀, the Raman spectrum showed 12 obvious characteristic peaks at 258.6, 701.4, 737.7, 770.6, 1061.1, 1184.0, 1229.2, 1332.8, 1368.0, 1447.2, 1513.6 and 1566.1 cm⁻¹.⁷⁷ The strongest peak was at about 1566.1 cm⁻¹. For ¹³C₅C₆₅, five incorporated ¹³C atoms did not alter the Raman vibrational modes of C₇₀, with no new characteristic peaks, and only caused the characteristic peaks to shift down 3–4 wavenumbers (Table 3). In view of the lower molecular symmetry of C₇₀, a few introduced ¹³C atoms may be not be enough to cause strong alterations in the molecular vibration spectra of high carbon cage-based fullerenes. More incorporated atoms exacerbated the change of symmetry, reducing the degeneracy and potentially triggering some new characteristic peaks. But for the lower molecular symmetry of high carbon cage-based fullerenes, the ¹³C stable isotope effects were possibly relatively weak and would not easily alter or damage the intrinsic structure of the fullerenes. The IR and Raman spectra showed that the incorporated ¹³C did not change the overall structure of the fullerenes. But in our previous investigations, with increased amounts of ¹³C, the Raman spectrum of ¹³C-enriched C₆₀ shifted to lower wavenumbers, the Raman silent mode was activated and new vibrational peaks emerged.⁶⁹ These changes were due to the mass addition caused by the incorporation of ¹³C atoms which easily broke the high symmetry structure of C₆₀.

Fig. 5 Raman spectra of ¹³C₅C₆₅ and C₇₀.

Table 3 Displacement of the characteristic Raman peaks of C₇₀ and ¹³C₅C₆₅

	νRaman/cm^−1
C70	258.6
	701.4	737.7	770.6
	1061.1	1184.0	1229.2	1332.8	1368.0
	1447.2	1513.6	1566.1
^13C5C65	255.4
	698.3	733.0	765.9
	1055.0	1180.0	1223.3	1328.4	1362.2
	1441.4	1507.8	1563.3

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Electrochemical analysis

Electrochemical measurements are the main methods used to characterize the electronic properties of fullerenes and their derivatives. Fig. 6 shows the electrochemical properties of the purified C₇₀ and ¹³C-enriched C₇₀ samples. Three reversible electron reduction waves were observed, which verified the good electron withdrawing ability of C₇₀ molecules. These results were similar to previous reports.^78–81 The cyclic voltammogram of ¹³C-enriched C₇₀ showed clearly a positive shift relative to unlabelled C₇₀. Incorporation of the stable isotope ¹³C into the fullerene molecular structure significantly influenced the half-wave potentials of C₇₀. The electron affinity of ¹³C-enriched C₇₀ should be weaker than that of unlabeled C₇₀ in the redox process. These diversities may be due to the addition of ¹³C and caused the HOMO–LUMO energy difference between C₇₀ and ¹³C-enriched C₇₀, altered the C₇₀ molecular strain and reduced the degree of conjugation of the molecules. This isotope effect on the electrochemical properties of fullerene C₇₀ has not been reported yet. These data again demonstrated that the ¹³C atom labels break the mass distribution and change the electron configuration of the C₇₀ molecule.

Fig. 6 Cyclic voltammograms of ¹³C₅C₆₅ and C₇₀.

Conclusions

In the present work, ¹³C-enriched large carbon cage-based fullerenes were synthesized on a large scale by an arc discharge method. The direct stable isotope ¹³C labeling on the skeleton of the fullerenes retained their intrinsic structures. The stable isotope atoms were randomly distributed in the carbon cage. The ¹³C labelling amounts of C₇₀ were higher than that of natural abundance, which greatly improved the ¹³C detection signal strength in IRMS, and also enhanced the signal strength and sensitivity of the fullerenes for CNMR. MS spectra showed that the isotopic effects fitted with a Poisson distribution. In the arc-discharge experiment, the ¹³C stable isotopic effects seemed to be weaker with increasing size of the fullerene carbon cages, especially for their molecular vibration spectra. Simultaneously, some ¹³C-enriched high carbon fullerenes (such as ¹³C-C₇₆, C₈₂ and C₉₆ etc.) also existed in the arc-discharge. The mechanism of fullerene formation in the arc-discharge process was speculated by contrasting ¹³C labelling amounts and isotopic effects between ¹³C-enriched C₇₀ and those of ¹³C-enriched C₆₀. The skeleton ¹³C-labeling technique would elucidate fully the formation mechanism of fullerenes and other carbon materials. Relying on the diversity of spectroscopic methods and IRMS, ¹³C stable isotope labeling methods provide a convenient and safer way to reflect the real properties of C₇₀ in biological systems. However, due to the expensive pure ¹³C powder the manufacturing cost of ¹³C-enriched C₇₀ is very high. It is also worth noting that the amount needed for a tracing experiment in vivo is small, usually less than 50 mg, and the detection sensitivity of IRMS for ¹³C is very high.^48–50 Therefore, the introduction of a stable isotope labeling technique makes the biomedical effect research of fullerene nanomaterials safer and more effective compared with radioactive markers.^82–86 We have confidence that the stable isotope labeling provides a new analytical method of in vivo quantitative and intrinsic detection for carbon nanomaterials and can be widely used in the nanomedical fields of fullerene-based nanomaterials and other carbon materials.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 11475194, 11005116 and 21307101), Beijing Natural Science Foundation (2152038) and the National Basic Research Program of China (973 program: 2012CB932601 and 2013CB932703). We thank Mrs Ping Liang at the Analytical and Testing Center, Huazhong University of Science & Technology, for the CNMR analyses.

Notes and references

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Footnote

† C. L. Wang and L. F. Ruan contributed equally.

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