Introduction

Fullerenes have been investigated for applications such as novel pharmaceuticals and cosmetics.1 C60 fullerenes and their derivatives have been reported to possess activity as enzyme inhibitors,2 antivirals,3 DNA scission agents,4 radical quenchers5 and photodynamic therapy (PDT) agents.6 In addition, C60 and C70 fullerenes have been proposed as photosensitizers for PDT to eliminate cancerous tissue because fullerenes can efficiently generate active oxygen upon visible light irradiation.7 However, biological applications of fullerenes have been limited because the solubility of fullerenes in water is extremely low.8 Various C60 fullerene derivatives have been synthesized to improve water solubility,9 and methods to improve the solubilization of fullerenes in water have been studied.10, 11 In addition, solubilization of C60 in water has been investigated using inclusion complexes composed of C60 and host molecules, such as cyclodextrin and calixarene,12, 13, 14, 15 micelles,16 liposomes17 and poly(N-vinylpyrrolidone) (PVP).18, 19 In particular, PVP can form water-soluble complexes with fullerenes simply by grinding them together in a mortar, which results in charge transfer interactions. Complexation with PVP can better solubilize fullerenes at high concentrations compared with other methods.

Successful PDT requires the accumulation of the photosensitizer only around cancerous tissue. Vascular endothelium in cancerous tissue contains pores on the order of tens to hundreds of nanometers (nm), which are not observed in healthy tissue. If the size of a drug is less than several nanometers, then the drug is removed quickly through kidney clearance. If the size of a drug is >400 nm, then it is removed quickly by macrophages. However, a drug with a size on the order of tens to a hundred nanometers can accumulate in cancerous tissue through an enhanced permeation and retention effect.20 Drugs within this size range can remain in cancerous tissue for a long period because the lymphatic vessels around cancerous tissue are undeveloped with an incomplete collection mechanism. Thus PDT photosensitizers accumulated around cancerous tissue can damage and kill only cancer cells by the active oxygen generated upon light irradiation.

The vinyl monomer, 2-methacryloyloxyethyl phosphorylcholine (MPC), has a pendant phosphorylcholine group, which is ubiquitous on the surface of cell membranes. The phosphorylcholine group has a betaine structure and a hydrophilic nature.21, 22 The MPC homopolymer (Figure 1a) PMPCn is well studied as a biomaterial because it shows inhibition of both blood coagulation and immunoresponses.23

Figure 1
figure 1

(a) Chemical structure of PMPCn. (b) Conceptual illustration of the preparation of the fullerene/PMPCn complex. (c) Application of the fullerene/PMPCn complex for PDT. PMPC, poly(2-(methacryloyloxy)ethyl phosphorylcholine); PDT, photodynamic therapy. A full color version of this figure is available at the Polymer Journal online.

Full size image

In this study, PMPCn, with a well-controlled structure, was prepared via reversible addition–fragmentation chain transfer (RAFT) radical polymerization. Fullerene and PMPCn powders were mixed using an agate mortar to form water-soluble fullerene/PMPCn complexes. The complexation of fullerenes with PMPCn can solubilize fullerenes in water (Figure 1b). The generation of singlet oxygen from the complexes upon visible light irradiation was confirmed by photooxidation of 9,10-anthracendipropionic acid (ADPA). Use of the complex in PDT was attempted because the water-soluble complexes are biocompatible owing to the surface PMPCn chains (Figure 1c).

Experimental procedure

Materials

MPC was purchased from NOF Corp. (Tokyo, Japan), which was produced using a previously reported method.21 4,4′-Azobis(4-cyanovaleric acid) (V-501, 98%) and poly(acrylic acid) (PAA, molecular weight (Mw)=2.50 × 104) from Wako Pure Chemical (Osaka, Japan) were used as received. 4-Cyanopentanoic acid dithiobenzoate (CPD) was synthesized according to previously reported methods.24 Methanol was dried over 4-Å molecular sieves and purified by distillation. The fullerenes (C60: 98% and C70: ⩾99%) from Sigma-Aldrich (St Louis, MO, USA) were used as received without further purification. Poly(methacrylic acid) (PMA) was prepared by neutralization of poly(sodium methacrylate) (Mw=9.50 × 103) from Sigma-Aldrich (St Louis, MO, USA). ADPA was prepared according to a method previously reported.25 Water was purified using a Millipore (Billerica, MA, USA) Milli-Q system.

Preparation of polyMPC (PMPCn)

The MPC homopolymer was synthesized according to a modified version of previously reported methods.23 MPC (10.8 g, 36.7 mmol), CPD (94.7 mg, 0.339 mmol) and V-501 (47.6 mg, 0.170 mmol) were dissolved in a mixed solvent (36.7 ml) of water and methanol (4/1 v/v). The solution was degassed by purging with Ar gas for 30 min. Polymerization was performed at 70 °C for 6 h. After the reaction, 1H nuclear magnetic resonance (NMR) data indicated that the conversion was 100%. The polymerization mixture was dialyzed against pure water for 2 days.26 PMPC113 was recovered by a freeze-drying technique (9.30 g, 85.0%). The number-average degree of polymerization (DP) and the number-average molecular weight estimated from NMR (Mn(NMR)) for PMPC113 were 113 and 3.36 × 104, respectively. The number-average molecular weight (Mn(GPC)) and molecular weight distribution (Mw/Mn) estimated from gel-permeation chromatography (GPC) were 1.93 × 104 and 1.17, respectively. Low-molecular-weight PMPC64 (DP=64, Mn(NMR)=1.89 × 104, Mw/Mn=1.23) and high-molecular-weight PMPC362 (DP=362, Mn(NMR)=1.07 × 105, Mw/Mn=1.97) were prepared using methods similar to those described above.

Preparation of fullerene/PMPCn complexes

A typical preparation of a fullerene/PMPC113 complex is as follows: PMPC113 (10 mg, 2.98 × 10−4 mmol, Mn(NMR)=3.36 × 104, Mw/Mn=1.17) and C60 (11 mg, 15.3 × 10−3 mmol) powders were mixed in an agate mortar and then vigorously mulled for 30 min. Water (10 ml) was added to the powder mixture. The final polymer concentration (Cp) was adjusted to 1.0 g l−1 by adding water. Insoluble material was removed by centrifugation at 6000 r.p.m. for 1 h. All sample solutions were filtered with a 0.45-μm membrane filter. Other concentrations of aqueous C60 and C70 solutions were prepared using methods similar to those described above.

Measurements

1H NMR spectra were obtained with a Bruker BioSpin (Billerica, MA, USA) DRX-500 NMR spectrometer operating at 500 MHz. The sample solutions for 1H NMR measurements were prepared using D2O. The GPC measurements were performed using a refractive index detector equipped with a Shodex (Tokyo, Japan) 7.0-μm bead size GF-7 M HQ column (exclusion limit ~107) operated at 40 °C under a flow rate of 0.6 ml min−1. A phosphate buffer (pH 9) containing 10 vol% acetonitrile was used as the eluent. The Mn(GPC) and Mw/Mn values were calibrated using standard sodium poly(styrene sulfonate) samples. Ultraviolet-visible (UV-vis) absorption spectra were recorded on a Jasco (Tokyo, Japan) V-630 UV-vis BIO spectrophotometer with a 1.0-cm path length quartz cell. Dynamic light scattering (DLS) measurements were performed using a Malvern (Worcestershire, UK) Zetasizer nano ZS at 25 °C. A He–Ne laser (4.0 mW at 632.8 nm) was used as the light source. Sample solutions were filtered using a 0.45-μm pore size membrane filter. The data were analyzed by the Malvern Zetasizer 6.20 software. Static light scattering (SLS) measurements were performed using an Otsuka Electronics Photal (Osaka, Japan) DLS-7000HL light scattering spectrometer. A He-Ne laser (10.0 mW at 632.8 nm) was used as the light source. Sample solutions for light scattering measurements were filtered with a 0.45-μm pore size membrane filter. The weight-average molecular weight (Mw), the z-average radius of gyration (Rg), and the second virial coefficient (A2) values were estimated from the following relationship:

where Rθ is the difference between the Rayleigh ratio of the solution and that of the solvent, K=4π2n2(dn/dCp)2/NAλ4, where dn/dCp is the refractive index increment against Cp, NA is Avogadro’s number and q is the magnitude of the scattering vector. The q value was calculated from q=(4πn/λ)sin(θ/2), where n is the refractive index of the solvent, λ is the wavelength of light source (=632.8 nm) and θ is the scattering angle. By measuring Rθ for a set of Cp and θ, the values of Mw, Rg and A2 were estimated from Zimm plots. The known Rayleigh ratio of toluene was used to calibrate the instrument. The values of dn/dCp at 633 nm were determined using an Otsuka Electronics Photal DRM-3000 differential refractometer. The dn/dCp values for C60/PMPC113 and C70/PMPC113 were 0.722 and 0.833 ml g−1, respectively. Small-angle X-ray scattering (SAXS) measurements were performed on a BL40B2 beamline at SPring-8, Hyogo, Japan. A 30 × 30 cm2 imaging plate (Rigaku R-AXIS VII, Tokyo, Japan) detector was used and was placed 4.0 and 2.0 m away from the sample. The wavelength of the incident beam (λ) was 0.10 nm. This setup provided a q range of 0.04−3.5 nm−1, where q is the magnitude of the scattering vector defined by q=(4πn/λ)sinθ with a scattering angle of 2θ. For the SAXS measurements, we used a vacuum sample chamber to remove the effects of the background from the window material and air. The X-ray transmittance of the samples was measured with an ion chamber located in front of the sample and a Si photodiode (Hamamatsu Photonics S8193, Shizuoka, Japan) placed after the sample. Detailed experimental procedures are reported elsewhere.27, 28 Transmission electron microscopy (TEM) was performed using a JEOL (Tokyo, Japan) JEM-2100 at an accelerating voltage of 200 kV. Samples for TEM observations were prepared by placing one drop of an aqueous solution of the fullerene/PMPC113 complex on a copper grid coated with support films. The samples were stained with phosphotungstic acid.

Generation of singlet oxygen

Photoirradiation was performed using an Asahi Spectra (Tokyo, Japan) Max-301 with light from a 300-W Xe lamp passing through a 420-nm cutoff filter. The light intensity was 6.5 mW cm−2 at 420 nm. A quartz cuvette with a 1-cm pass length was used. ADPA bleaching experiments were performed to confirm that singlet oxygen was generated by fullerene/PMPC113 complexes in water, where ADPA was generated as a singlet oxygen acceptor. Aqueous solutions of ADPA and fullerene/PMPC113 complexes were mixed, and the combined solution was irradiated at ⩾420 nm. The reaction was monitored by recording a decrease in the intensity of the absorption peak at 400 nm owing to ADPA as a function of the irradiation time.

Results and discussion

Characterization of PMPCn

MPC was polymerized via a RAFT-controlled radical polymerization method using CPD; the MPC conversion was 100%. The DP and Mn(NMR) values estimated from NMR for PMPC113 were 113 and 3.36 × 104, respectively (Table 1). The Mn(GPC) and Mw/Mn values estimated from GPC were 1.93 × 104 and 1.17, respectively. Low-molecular-weight (Mn(NMR)=1.89 × 104) and high-molecular-weight PMPCn (Mn(NMR)=1.07 × 105) were also prepared. After polymerization, the percentage of conversion (x) was monitored using 1H NMR before purification, which can be estimated by comparing the integral intensity ratios between vinyl protons and pendant methylene protons. The value of Mn(theoretical) was calculated using the following equation:

Table 1 Molecular characteristics of PMPCn
Full size table

where [MPC]0 is the initial molar concentration of MPC, [CPD]0 is the initial molar concentration of CPD, MWMPC is the molecular weight of MPC and MWCPD is the molecular weight of CPD.

Proton NMR spectra for PMPC113 in D2O were obtained (Supplementary Figure S1). Signals in the 7.45–7.95 p.p.m. region were attributed to the dithiobenzoate group. The DP of PMPC113 was calculated as 113 from the area intensity ratio of peaks attributed to pendant methylene protons at 3.65 p.p.m. and terminal phenyl protons. The value of Mn(NMR) for PMPCn can be calculated from the corresponding DP, which is close to Mn(theoretical). The GPC peak for PMPC113 was unimodal (Supplementary Figure S2). The Mw/Mn value for PMPC113 was 1.17, suggesting that PMPC113 had a well-controlled structure. The Mn(GPC) value was an estimated value because poly(sodium p-styrenesulfonate) was used as a standard for the calibration.

Preparation and characterization of fullerene/PMPCn complexes

The sample preparation method is important for the solubilization of fullerenes in water using PMPCn. Fullerene and PMPCn powders were ground with a mortar and pestle for 30 min to achieve homogeneity.29 The relationship between the amount of fullerene solubilized in water using PMPCn and the duration of grinding was determined. The amount of fullerene solubilized was not increased by grinding for >30 min. Therefore, the grinding time was kept constant at 30 min to prepare the fullerene/PMPCn complexes. Insoluble matter was removed by centrifugation. All sample solutions were filtered with a 0.45-μm membrane filter.

To measure UV-vis absorption spectra for C60/PMPC113 and C70/PMPC113 complexes in water with varying fullerene concentrations, the sample solutions were diluted with water because the absorbance from the original aqueous complex solutions was too high to measure using a UV-vis absorption spectrometer (Figure 2). The original absorbance was calculated from the absorption spectra and the dilution ratio. The C60/PMPC113 complex indicated C60 absorption peaks at 215, 265 and 340 nm, which increased until [C60]⩽1.1 g l−1 while increasing the C60 feed concentration. The C70 absorption peaks at 215, 240 and 384 nm for the C70/PMPC113 complex increased until [C70]⩽0.5 g l−1 while increasing the C70 feed concentration. These absorption peaks were similar to those for C60 and C70 in organic solvents.16 These observations indicate that PMPCn can solubilize C60 and C70 in water.

Figure 2
figure 2

UV-vis adsorption spectra for (a) C60/PMPC113 and (b) C70/PMPC113 complexes at various fullerene concentrations in water. The original solutions were diluted to 1/100 because the absorbance was too high. PMPC, poly(2-(methacryloyloxy)ethyl phosphorylcholine); UV-vis, ultraviolet-visible. A full color version of this figure is available at the Polymer Journal online.

Full size image

The relationship between the feed amount of fullerene and the amount of fullerene dissolved in water using PMPC113 is shown in Figure 3. The solubilized amounts were calculated from the absorbance and extinction coefficients for C60 (68.0 Lg−1 cm−1 at 340 nm) and C70 (40.4 Lg−1 cm−1 at 384 nm).18 The maximum concentrations of C60 and C70 in water using PMPC113 at Cp=1 g l−1 were 0.67 and 0.49 g l−1, respectively. The solubilized amounts of fullerenes using PMPC113 were higher than those in previous reports. The reported concentration of C60 in water using surfactants, such as lecithin, was 0.012 g l−1.16 Yamakoshi et al.18 reported that solubilized C60 and C70 concentrations using PVP in water were 0.4 and 0.2 g l−1, respectively.

Figure 3
figure 3

Plots of solubilized amounts of C60 (○) and C70 (△) as a function of feed concentration of C60 or C70. A full color version of this figure is available at Polymer Journal online.

Full size image

Although the pendant phosphorylcholine groups in PMPCn are hydrophilic, the main chain of methacrylate is slightly more hydrophobic than the pendant group. The methacrylate main chain in PMPCn may interact with hydrophobic fullerene. To confirm hydrophobic interactions between the main chain and fullerene, solubilization tests for C60 in water were performed using commercially available PMA (Mw=9.50 × 103) and PAA (Mw=2.50 × 104) powders. PMA at Cp=1 g l−1 dissolved C60 in water with [C60]=0.37 g l−1. In contrast, PAA could not dissolve C60 in water. These results suggest that a methacrylate-type main chain structure may be extremely important for the formation of a fullerene/PMPCn complex. The amount of fullerene solubilized by PMA was less than that by PMPCn. Thus the solubilization of fullerenes may require hydrophobic interactions between the methacrylate polymer backbone and fullerene as well as interactions between the pendant phosphorylcholine groups in PMPCn and fullerene. To confirm the influence of the Mw of PMPCn, solubilization of C60 in water was performed using PMPCn with a low (Mn(NMR)=1.89 × 104) and a high (Mn(NMR)=1.07 × 105) Mw. The maximum concentrations of C60 in water using PMPC64 and PMPC362 at Cp=1 g l−1 were 0.76 and 0.44 g l−1, respectively. The maximum C60 concentrations in water increased as the Mw of PMPCn decreased. For every polymer chain, the number of solubilized C60 molecules can be calculated from the molar concentrations of polymer and solubilized C60. The number of C60 molecules that could be solubilized by a single polymer chain of PMPC64, PMPC113, PMPC362 and PMA was 20, 31, 65 and 7, respectively.

The stability of the complex during dilution with water was studied. The hydrodynamic radius (Rh) values for the complexes were plotted as a function of Cp (Figure 4). The Rh values for the complexes were kept constant and were independent of Cp in the region of 1–0.05 g l−1. The complexes were stable even at 0.05 g l−1upon dilution of Cp. When Cp was diluted to a concentration <0.05 g l−1, DLS data could not be obtained owing to low scattering intensity.

Figure 4
figure 4

Rh of C60/PMPC113 (○) and C70/PMPC113 complexes (△) as a function of Cp. Cp, polymer concentration; PMPC, poly(2-(methacryloyloxy)ethyl phosphorylcholine); Rh, hydrodynamic radius. A full color version of this figure is available at the Polymer Journal online.

Full size image

To characterize the complexes in more detail, SLS measurements were performed for C60/PMPC113 and C70/PMPC113 complexes in water (Supplementary Figure S3). The results from light scattering measurements are summarized in Table 2. The values of Mw and Rg for the C60/PMPC113 complex were larger than were those for C70/PMPC113. The A2 values for C60/PMPC113 and C70/PMPC113 were 1.19 × 10−3 and 2.77 × 10−3 cm3·mol g−2, respectively. The complex using C60 had a small A2, which indicates that the solubility of C60/PMPC113 in water was lower than that of C70/PMPC113.30, 31 The Rg/Rh value is related to the shape and polydispersity of aggregates in solution. The theoretical Rg/Rh values for rigid hard spheres and spherical aggregates are 0.78 and 1.0, respectively.32, 33, 34, 35 The Rg/Rh values for random coil and ellipsoidal aggregates are 1.3–1.5. Thread-like and low-density aggregates with a high polydispersity index indicate larger Rg/Rh values. The shapes of the fullerene/PMPC113 complexes may be spherical because the Rg/Rh values for the complexes were close to 1. The densities of C60/PMPC113 and C70/PMPC113 complexes calculated from the apparent Mw and Rg values estimated from SLS were 0.103 and 0.064 g cm−3, respectively. The maximum number of fullerenes for every PMPC113 chain was calculated from the molar ratio of PMPC113 and fullerene in water using UV-vis absorption data. The number of C60 and C70 in one PMPC113 polymer chain was 31 and 20, respectively. The aggregation number (Nagg) for the complex was calculated from the apparent Mw for the complex and the Mw of a single polymer chain with the accompanying fullerenes. The Nagg was defined as the total number of PMPC113 chains in one complex. The Nagg values for C60/PMPC113 and C70/PMPC113 complexes were 96 and 40, respectively. The number of C60 and C70 fullerenes included in one complex was 2995 and 784, respectively.

Table 2 Dynamic and static light scattering data for fullerene/PMPC113 complexes in water at 25 °C
Full size table

To confirm the structure of the complex, we performed SAXS measurements (Figure 5). The Rg values for PMPC/C60 and PMPC/C70 were 18 and 20 nm, respectively, as obtained from Guinier plots. These values are smaller than those obtained from SLS measurements. As X-rays scatter owing to nanoscale electron density inhomogeneities within samples, they effectively highlight the clustered fullerene parts in the complex.36 The Rg of C70/PMPC was larger than that of C60/PMPC, as estimated from SAXS measurements. This observation suggests that the aggregate formed from C70 in the complex was swollen owing to incorporation of PMPC chains and water into the aggregate.

Figure 5
figure 5

(a) SAXS profiles and (b) Guinier plots for C60/PMPC113 and C70/PMPC113 complexes in water. a: shift factor, q: the magnitude of the scattering vector and I(q): scattering intensity.

Full size image

TEM measurements were performed to confirm the size and shape of the fullerene/PMPC113 complex (Figure 6). The C60/PMPC113 and C70/PMPC113 complexes had a distorted spherical shape. The average diameters estimated from TEM images for C60/PMPC113 and C70/PMPC113 complexes were 29 and 24 nm, respectively. The sizes estimated from TEM were slightly smaller than were those estimated from light scattering measurements because the complexes may shrink during the drying process while preparing TEM samples.

Figure 6
figure 6

TEM images for (a) C60/PMPC113 and (b) C70/PMPC113 complexes. PMPC, poly(2-(methacryloyloxy)ethyl phosphorylcholine); TEM, transmission electron microscopy.

Full size image

Generation of singlet oxygen

Fullerenes can be excited by light, and the energy is subsequently transferred to ground-state oxygen to efficiently generate singlet oxygen. The photooxidation of ADPA by singlet oxygen can be applied for the detection of generated singlet oxygen atoms using a photosensitizer (Scheme 1).25 To confirm the generation of singlet oxygen atoms from fullerene/PMPCn complexes upon visible light irradiation, ADPA was used to detect the singlet oxygen atoms in water. Photooxidation of ADPA to generate ADPA endoperoxide by singlet oxygen is indicated by observing a decrease in the UV-vis absorption peaks of ADPA. Changes in the UV-vis absorption spectra of ADPA were measured in the presence of fullerene/PMPCn complexes in water upon visible light irradiation at ⩾420 nm, as shown in Figure 7. The polymer concentration was diluted to 0.021 g l−1 in water because the absorbance of fullerenes was too high for the original solutions at Cp=1 g l−1. The concentrations of C60 and C70 were 0.0141 and 0.0103 g l−1, respectively. The concentrations of ADPA used in the C60 and C70 experiments were 0.0377 and 0.0439 g l−1, respectively. The UV-vis spectra were calibrated by subtracting the fullerene absorption. The absorbance of ADPA in the presence of the complex decreased with increasing irradiation time. After 180 min of irradiation, the absorbance values at 399 nm of ADPA in the presence of C60/PMPC113 and C70/PMPC113 complexes were 0.186 and 0.160, respectively. This observation indicates that C70/PMPC113 can oxidize ADPA more efficiently than C60/PMPC113. The amounts of singlet oxygen generated from C70/PMPC113 were higher than those from C60/PMPC113 by light irradiation above 420 nm, presumably because the absorption wavelength of C70/PMPC113 was longer than that of C60/PMPC113.

Figure 7
figure 7

Changes in UV-vis absorption spectra of ADPA in the presence of (a) C60/PMPC113 and (b) C70/PMPC113 complexes in water upon visible light irradiation (⩾420 nm). Fullerene absorptions were subtracted from the original spectra for calibration. Irradiation times are indicated in the figure. ADPA, 9,10-anthracendipropionic acid, PMPC, poly(2-(methacryloyloxy)ethyl phosphorylcholine); UV-vis, ultraviolet-visible. A full color version of this figure is available at the Polymer Journal online.

Full size image

Conclusions

Hydrophilic and biocompatible PMPCn with a well-controlled structure was prepared via RAFT-controlled radical polymerization. Fullerene/PMPCn complexes were prepared by mixing fullerene and PMPCn powders. Fullerenes could be solubilized in water using PMPCn with a higher concentration than those reported previously. Hydrophobic interactions of the main chain with fullerene and interactions between the pendant phosphorylcholine and fullerene may be important for fullerene solubilization. The Rh values for C60/PMPC113 and C70/PMPC113 complexes were 25.1 and 23.2 nm, respectively. The number of C60 and C70 fullerenes in one complex was 2995 and 784, respectively. Singlet oxygen was generated from fullerene/PMPCn complexes upon visible light irradiation >420 nm. These fullerene/PMPCn complexes are promising candidates for application in PDT.

scheme 1

Photooxidation of ADPA by singlet oxygen. ADPA, 9,10-anthracendipropionic acid.