Introduction

Conventional cyclodextrins (CDs) are cyclic oligosaccharides consisting of 6 (α-CD), 7 (β-CD), and 8 (γ-CD) d-glucose units linked by α-1,4 bonds. The structure of CD molecules resembles truncated cones with the secondary hydroxyl groups located at the wider edge of the ring and the primary groups on the narrower edge. Hydrogen atoms are directed to the inner part of the ring, resulting in a hydrophobic cavity along with a hydrophilic character outside the ring. Hence, CDs are soluble in water and at the same time may accommodate a broad range of hydrophobic species inside the cavity with formation of inclusion complexes. This property accounts for the great interest in CDs and it was shown that complex formation can be improved by chemical modifications of native CDs [1]. On the other hand, there have been few papers concerning large-ring CDs (LR-CDs) composed of more than nine α-1,4-linked d-glucose units, except for a paper by French et al. in 1965 which provided the first definitive evidence for the existence of such molecules. LR-CDs with 9, 10, 11, 12, and 13 d-glucose units were named δ-, ε-, ζ-, η-, and θ-CD, respectively [2]. However, due to low yields and difficulties in their purification, they have not been studied in detail. We have focused on LR-CDs for several years and have previously reported on the isolation and purification methods, physicochemical properties, and complex-forming ability of LR-CDs with 9–50 d-glucose units [3–6]. We have already determined the crystal structures for ε- and ι-CD [7, 8]. Most importantly, we have established an isolation and purification method to obtain relatively large amounts of δ-CD [9, 10] and have characterized its physicochemical properties and complex-forming abilities with a number of guest compounds [11–13].

The fullerene family, especially C60, has very appealing photo-, electro-, and physico-chemical properties, which can be exploited in many different biological fields [14]. However, the predominant hydrophobic character of fullerenes causes low solubility in polar media, such as water. Several groups reported successful techniques to overcome this problem that relied on embedding C60 in α-, β-, and γ-CD, respectively [15–17]. In addition, a C60/γ-CD complex exhibits significant inhibitory effects on the oxidative discoloration of β-carotene in scavenging harmful peroxyl radicals and reactive oxygen species [18]. Therefore, water-soluble fullerene can be expected as a new potential antioxidant tool in pharmaceutical and cosmetic fields.

Previously, we have reported that C70, a member of the fullerene family, could be solubilized in water by complexation with δ-CD [19, 20]. It was considered that C70 was better fitted to δ-CD than other conventional CDs since δ-CD has a larger cavity.

In this paper, the preparation method of the inclusion complex of C60 with δ-CD using a ball-milling method based on a solid–solid mechanochemical reaction is reported. The inclusion complex was characterized by UV–VIS spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and fast atom bombardment mass spectrometry (FAB-MS) in both the solution and solid state. Moreover, phase solubility studies were performed, which confirmed that complexation with δ-CD, including pretreatment by ball-milling, actually enhanced the C60 solubility and that the stoichiometric ratio between C60 and δ-CD in aqueous solution was 1:2 (C60:δ-CD).

Materials and methods

Materials

δ-CD in excess of 98% purity was prepared by the method described previously [9]. γ-CD was obtained from Wacker Chemicals East Asia Ltd. (Tokyo, Japan). δ-CD and γ-CD were used after recrystallization from water. C60 was purchased from Term USA (Berkeley, CA, USA), and purity of C60 was greater than 99%. All other chemicals and solvents were commercial grade and used without further purification. Milli-Q water was used in all experiments.

Instruments

A lightproof ball mill (model V-1M; Irie Shokai Co., Ltd., Tokyo, Japan) was used. A pot’s volume was 50 mL and inside diameter was 35 mm. The enclosed glass balls had about 7 mm diameter. Powder X-ray diffraction (XRD) was recorded using an RINT 1400 X-ray diffractometer (Rigaku Co., LTD., Tokyo, Japan). X-Ray measurement was carried out at 1°/min from 4° to 40° of the diffraction angle at 60 kV and 200 mA at room temperature. UV–VIS spectrometry was measured using a Ubest-30 Double Beam spectrophotometer (JASCO Co., Ltd., Tokyo, Japan). FAB-MS was performed in negative ion mode by a JEOL SX-102A mass spectrometer (JEOL, Tokyo, Japan) with a Magic Bullet matrix and acceleration voltage of −6 kV. 1H- and 13C-NMR spectra at 500 MHz were taken on a JNM-LA500 spectrometer (JEOL, Tokyo, Japan) at 50 °C.

Determination of the time for a ground mixture of C60 with δ-CD

A mixture of 16.9 mg of C60 (2.4 × 10−5 mol) and 35.0 mg of δ-CD (2.4 × 10−5 mol) was ball-milled at room temperature. The mixture was measured by XRD after ball-milling for 6, 12, 24, and 48 h.

Preparation of the complexes of C60 with δ-CD in aqueous solution and analysis by spectrophotometry

Initially, a mixture of 16.9 mg of C60 (2.4 × 10−5 mol) and 35.0 mg of δ-CD (2.4 × 10−5 mol) was ball-milled for 24 h at room temperature. After adding 25 mL of water, the solution was incubated at 25 °C for about 3 days. The suspension was centrifuged at 25 °C and 10,000 rpm (maximum gravity approximately 12,000×g) for 1 h. The supernatant was examined by UV–VIS spectroscopy at a wavelength of 338.5 nm. The C60/γ-CD system was studied in the same way as that of C60/δ-CD system.

Characterization of the C60/δ-CD system by NMR spectrometry

A mixture of 16.9 mg of C60 (2.4 × 10−5 mol) and 175.0 mg of δ-CD (1.2 × 10−4 mol) was ball-milled, incubated and centrifuged as described in the previous section. Then, water in the supernatant was removed by freeze-drying. The solid sample of C60/δ-CD system was dissolved in 99.95% deuterium oxide (D2O). Chemical shifts were reported in δ-units (ppm) downfield from the signal of external tetramethylsilane.

Determination of the stoichiometry of the C60/δ-CD system by FAB-MS

FAB-MS analysis of the C60/δ-CD system was performed after freeze drying to remove water from the aqueous samples, in a similar manner as the sample preparation for UV–VIS spectrometry.

Phase solubility diagram of C60 with δ-CD

The phase solubility study was carried out according to Higuchi and Connors [21]. Excess amounts (10 mg) of C60 were added to 0.01–0.05 M δ-CD aqueous solutions. The solutions were dissolved under sonication and incubated at 10 and 25 °C. After equilibrium was attained (approximately 4 days), the solution was filtered through a membrane (0.45 μm, KURABO INDUSTRIES LTD., Osaka, Japan). After adding toluene (2 mL) to the filtrate (2 mL), the suspension was centrifuged at 20 °C, 3,000 rpm for 10 min. This procedure was repeated 10 times. Through the extraction process, C60 was completely transferred into the organic phase. The extract was evaporated to dryness under reduced pressure and re-dissolved in 10 mL toluene. Each 10 μL toluene solution was analyzed by HPLC. HPLC analysis was performed on a LC-6A liquid chromatograph (Shimadzu Corporation, Kyoto, Japan) using an Inertsil ODS-2 (150 × 4.6 mm i.d.) column (GL-Sciences Inc., Tokyo, Japan) with a SPD-6A UV detector at 260 nm (Shimadzu). Hexane/2-propanol (50:50 v/v) was used as the mobile phase (flow rate = 1 mL/min).

Results and discussion

Determination of the time taken to obtain a ground mixture of C60 with δ-CD

A cogrinding technique is widely used for reducing the particle size of a solid to enable dissolution of water-insoluble compounds in water since dissolution rate depends strongly on particle size. Reduced particle size is a consequence of strong grinding force on a solid and causes an increase in activation energy on the surface and distortion of the crystal lattice. Braun et al. [22] reported the preparation method to dissolve C60 in water by grinding between C60 and γ-CD. Hence, the theory and method of cogrinding were applied for the solubilization of C60 with δ-CD.

XRD patterns of δ-CD, C60 and a physical mixture of δ-CD and C60 (molar ratio 1:1) are presented in Fig. 1a. The pattern of the physical mixture could be interpreted as an approximate superposition of δ-CD and C60. It was revealed that no interaction occurred between δ-CD and C60 by physical mixing alone. The peak intensity of each line decreased with increasing grinding time and a broad pattern was observed after 6, 12, 24, and 48 h of grinding, as shown in Fig. 1b. The changes in XRD patterns upon grinding indicated a decrease in proportion of crystalline forms of δ-CD and C60, and no new diffraction peak appeared. This suggests that the crystalline structure of C60 and δ-CD were disrupted and that both C60 and δ-CD molecules were substantially converted into amorphous states, but did not form a crystalline complex. Moreover, the diffraction patterns did not alter after 24 h of grinding; suggesting adequate duration for obtaining a ground mixture of δ-CD and C60 under our experimental conditions.

Fig. 1
figure 1

XRD patterns of δ-CD, C60 and the C60/δ-CD system (physical mixture) (a) and effect of ball-milling time on C60/δ-CD system on the XRD patterns (b)

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UV–VIS spectrum of the C60/δ-CD system

The UV–VIS spectrum of the C60/δ-CD system is shown in Fig. 2a. The spectrum of the C60/δ-CD system was in agreement with that of C60 hexane solution [23, 24] and was similar to that of the C60/γ-CD complex in aqueous solution [17]. A maximum UV–VIS absorption (λmax) for this system was observed at 262 nm and the spectrum of the C60/δ-CD system in water was slightly red-shifted compared to those of the C60 hexane solution [23, 24] and the C60/γ-CD complex in aqueous solution [25]. This phenomenon could be explained by the intermolecular interaction between the cavity of δ-CD and the π-systems of C60. Furthermore, the C60/δ-CD system solution appeared yellowish-brown; similar to the coloration of C60 in 1,2-dimethoxy-benzene solution [26]. These results suggested that it was possible to dissolve C60 in water to allow formation of a complex between C60 and δ-CD.

Fig. 2
figure 2

UV–VIS spectra of C60/δ-CD complex (diluted 20 times) (a) and C60/γ-CD complex (diluted 10 times) (b) in aqueous solution

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Figure 2b shows the UV–VIS spectrum of the C60/γ-CD system under our experimental conditions and is consistent with spectra of the C60/δ-CD system and C60 in hexane solution. This result demonstrates that interactions between C60 and the γ-CD in water could be reproduced under our experimental conditions. However, the spectral intensity of the C60/γ-CD system was lower than that of the C60/δ-CD system and shows that the solubility of complexed C60 with γ-CD in water was lower than with that of δ-CD. The concentration of C60 in the C60/δ-CD system in water was found to be about 1.57 × 10−4 M using the UV absorption method; which was higher than the value of C60/γ-CD complex [17]. δ-CD would incorporate C60 more efficiently than γ-CD due to its larger cavity.

Characterization of C60/δ-CD system by NMR spectrometry

In order to clarify the interaction between δ-CD and C60 in aqueous solution, 1H-NMR and 13C-NMR spectra of the C60/δ-CD system were measured in D2O at 50 °C. When the sample was prepared at a molar ratio of 1:1 (C60:δ-CD), the 1H- and 13C-NMR spectra of the C60/δ-CD system were almost the same as that of δ-CD alone. It was considered that this sample did not give adequate solubility of C60 in D2O to be measured by NMR spectrometry. Hence, the sample was prepared at a molar ratio of 1:5 (C60:δ-CD).

From the 1H-NMR spectroscopy, the chemical shifts of the C60/δ-CD system in D2O were almost the same as that of δ-CD alone (data not shown). It is well known that protons at the H-5 and H-3 positions in d-glucose units are upfield shifted by the insertion of a guest compound in the inner hydrophobic space of the CD ring [27]. If the C60 molecule inserts into the inner space of the δ-CD molecule by inclusion complex formation, the protons H-5 and H-3 would be most influenced by the insertion. However, we did not observe this phenomenon. Similar observation of the 1H-NMR on C60/γ-CD system was reported by several groups [28–30].

The 13C-NMR spectrum of C60/δ-CD system is shown in Fig. 3. A signal at 144.4 ppm, which was close to that reported for C60 in organic solvents [31], and the other six signals derived from δ-CD were observed. The signals of all carbons moved an average of 0.2 ppm toward the lower field region than δ-CD alone (data not shown). Kanazawa et al. [29] reported similar observations in the C60/γ-CD system. They considered that the 13C-NMR changes indicated that a part of the C60 molecule intrudes into the inner space of the γ-CD and that rotation of the C60 molecule within the complex occurs rapidly on the NMR time scale. It seemed appropriate to consider our results in a similar way, that is, the result indicated the formation of a water-soluble supramolecular complex between δ-CD and C60.

Fig. 3
figure 3

13C-NMR spectrum of C60/δ-CD complex in D2O at 50 °C

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Determination of the stoichiometry of the C60/δ-CD system by FAB-MS

From UV–VIS and 13C-NMR measurements, it was suggested that an interaction between C60 and δ-CD had been revealed, however this was not verified by the stoichiometry of the C60/δ-CD system.

As a soft ionization technique, fast atom bombardment (FAB), electrospray ionization (ESI), and matrix assisted laser desorption/ionization (MALDI) mass spectrometry have been important tools in research of non-covalent complexes in recent years due to many advantages over other techniques [32]. High sensitivity, specificity, and speed are the proverbial virtues. Furthermore, FAB, ESI, and MALDI-MS can provide stoichiometric information for the complex directly and detect multiple components simultaneously.

Andersson et al. [33] reported that a complex consisting of two γ-CDs and one molecule of C60 was identified by negative fast atom bombardment/liquid secondary ion (FAB/LSI) mass spectroscopy. Here the use of FAB-MS for determination of the stoichiometric ratios of C60/δ-CD system is described.

The FAB-MS spectrum of the C60/δ-CD system is shown in Fig. 4. The peaks at m/z 721.1 and m/z 1459.3 correspond to the negative ions for C60 and δ-CD, respectively. In addition, the peak at m/z 2916.9 corresponds to the dimer δ-CD. Furthermore, a peak at m/z 3638.0 was observed and corresponds to a complex of two δ-CD and C60. This result suggested a stoichiometric ratio of the C60/δ-CD complex was 1:2 (C60:δ-CD).

Fig. 4
figure 4

FAB-MS spectrum of C60/δ-CD complex

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Phase solubility diagram of C60 with δ-CD

From the FAB-MS spectrum, the formation of the C60/δ-CD complex with a stoichiometry of 1:2 (C60:δ-CD) in solid state was identified. However, the stoichiometric ratio of the C60/δ-CD complex in aqueous solution had not been observed yet. In order to understand the mechanism of inclusion complex formation of C60 with δ-CD, the behavior of inclusion complex formations for C60 with δ-CD in aqueous solution was studied using the phase solubility method. Although attempts were made to develop a phase solubility diagram of the C60/δ-CD system, it could not clearly be classified as A or B type at 25 °C. It was suggested that the driving force of inclusion complex formation would decrease with increasing temperature due to the decrease in interactions, such as van der Waals and hydrophobic forces [34]. Thus, the phase solubility diagram of C60/δ-CD system was carried out at 10 °C (Fig. 5). The system showed typical Bs-type solubility curve, where the initial rising portions are followed by plateau regions and then the total C60 concentration decreases with the precipitation of complexes. From the diagram obtained, the stoichiometry of the complex was found to be 1:2 (C60:δ-CD), in agreement with the stoichiometry in solid state as suggested from the FAB-MS study. Furthermore, the solubility of C60 was about 1.3 × 10−5 M in this system in a preparation method without ball-milling. This value was lower than that with the ball-mill process (1.57 × 10−4 M). Therefore, the cogrinding with CD using ball-mill method could be powerful tool for solubilization of water-insoluble fullerenes into water.

Fig. 5
figure 5

Phase solubility diagram of C60/δ-CD system in water at 10 °C

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Conclusion

A successful dissolution of C60 in a water inclusion complex with δ-CD, an LR-CD composed of nine α-1,4-linked d-glucose units, has been achieved using a ground mixture preparation. A duration of 24 h was found to be adequate to obtain ground mixtures between δ-CD and C60. From the result of 13C-NMR spectrometry, an interaction between δ-CD and C60 in aqueous solution was revealed. Furthermore, FAB-MS data and a phase solubility diagram showed the formation of 1:2 complexes of the C60/δ-CD system in both the solid state and aqueous solution. The solubility of C60 in water was about 1.57 × 10−4 M using UV–VIS spectrometry, which was improved in comparison with that derived from the preparation method without ball-milling. These results showed that δ-CD was more suitable host molecule for C60 than conventional CDs and may show a potential of LR-CDs for dissolving fullerenes in water.