Applications of functionalized fullerenes in tumor theranostics.

Functionalized fullerenes with specific physicochemical properties have been developed for cancer diagnosis and therapy. Notably, metallofullerene is a new class of magnetic resonance imaging (MRI) contrast-enhancing agent, and may have promising applications for clinical diagnosis. Polyhydroxylated and carboxyl fullerenes have been applied to photoacoustic imaging. Moreover, in recent years, functionalized fullerenes have shown potential in tumor therapies, such as photodynamic therapy, photothermal treatment, radiotherapy and chemotherapeutics. Their antitumor effects may be associated with the modulation of oxidative stress, anti-angiogenesis, and immunostimulatory activity. While various types of novel nanoparticle agents have been exploited in tumor theranostics, their distribution, metabolism and toxicity in organisms have also been a source of concern among researchers. The present review summarizes the potential of fullerenes as tumor theranostics agents and their possible underlying mechanisms are discussed.

Introduction

Buckminsterfullerene (C60) is a carbon allotrope discovered in 1985 1. Since then, fullerenes have attracted much attention and been widely investigated. After the main problem of poor solubility was solved by chemical or supramolecular approaches (usually a chemical modification on the surface of the sphere), various functionalized fullerenes were synthesized to achieve promising results 2. For example, the empty interior cavity of the fullerene molecule makes it an ideal site for containment of a metal atom to form metallofullerene. This special structure of metallofullerene shows advantages compared to the metal itself or metal chelate complexes. One of their most important and novel electronic properties is the so-called intra-fullerene electron transfer from the encaged metal atom(s) to the fullerene cage(s), which has been suggested to use in MRI 3. Most likely due to its unique chemical structure, C60 possesses interesting photo-physical properties and generates reactive oxygen species (ROS) by exposure to visible light, making it a potentially strong agent for photodynamic therapy in biological systems 4.

The specific physicochemical properties of fullerenes promoted the development of tumor theranostic; consequently, functionalized fullerenes are currently under extensive investigation due to their potential application to tumor imaging and therapy (Table ).

Functionalized fullerenes for tumor imaging

Magnetic resonance imaging

Among many imaging strategies, such as optical imaging (OI), computed tomography (CT), MRI, ultrasound (US), and radionuclide imaging with positron emission tomography (PET) or single photon emission computed tomography (SPECT), MRI is a versatile, non-invasive medical diagnostic technique that can provide both physiological and anatomical information, while not requiring the use of ionizing radiation. In addition, in many applications it is often the most sensitive method available to depict soft tissues. Therefore, it has been extensively used for brain and central nervous system imaging, for assessing cardiac function and for detecting abnormal tissues such as tumors 5, 6. However, the difference between MR images of normal and abnormal tissues is often not large enough; therefore, specific exogenous contrast agents are often required to increase MR image quality and obtain higher resolution and sensitivity.

Paramagnetic metal ions, especially gadolinium (Gd3+), have been proposed as MR contrast agents. Conventional T1 contrast agents have been developed in the form of more stable and less toxic metallochelates, including gadolinium(III) diethyltriaminepentaacetic acid (Gd(III)-DTPA) and gadolinium-tetraazacyclododecanetetraacetic acid (Gd(III)-DOTA), which are currently marketed as Omniscan® and ProHance®, respectively 6, 7. However, for Gd3+ chelate complexes, the main limitation is the release of metal ions in vivo during metabolic processes and the subsequent toxicity. Since gadolinium-containing metallofullerenes were first used as excellent contrast agents for MRI with relatively high spatiotemporal resolution, they have attracted much attention for their potential application in clinical diagnosis. The Gd3+ ion was encapsulated in the fullerene cage, which is a structure that preserves the properties of the metal ion, avoids any leakage and thus prevents its dissociation in vivo 7. After specific chemical modifications, various Gd@Cn (n=60, 82) derivatives have been developed and widely explored (Figure ) 8, 9.

Gd@C82(OH)n has shown a water proton relaxivity twenty times higher than the commercially available Gd-DTPA. After intravenous (iv) administration of Gd@C82(OH)40 at a dose of 1/20 of the typical clinical dose of Gd-DTPA, a higher signal enhancement was measured in vivo (Figure ) 10. To achieve better bio-distribution, modification of the fullerene cages with biologically active groups has often been proposed. For example, the organic phosphonate modified Gd@C82O2(OH)16(C(PO3Et2)2)10 has exhibited high affinity to bone 11.

For a long time, the greatest obstacle to the practical application of Gd@C82 was its low production yield and requirement for potentially laborious HPLC purification. Recently, without the complicated process of purification, Grushko et al. tested the MRI efficiency of a mixture of gadolinium endometallofullerenes (with high content of more than 80 mol.%) and empty fullerenes, and found that the T1-weighted signal intensity was 15~30 times higher than the commercial “Gadovist” contrast agent 12.

After the Gd@C82 contrast agent was developed, another type of gadofullerene derivative, Gd@C60[C(COOH)2]10 was also found to possess a higher relaxivity (4.6 mM-1 s-1 at 20 MHz and 40 °C) compared to that of commercial Gd3+ chelate. It was used as an efficient cellular magnetic resonance imaging label in vitro and a 2.5-fold signal enhancement was shown in T1-weighted MRI of labeled cells compared to control cells. The cellular uptake characteristics of this gadofullerene were significant and nonspecific, and an excellent labeling efficiency (98-100%) was achieved without any adjunctive agent, compared to Gd@C82(OH)40. Furthermore, Gd@C60[C(COOH)2]10 was the first highly soluble, air-stable discrete molecule based on the more abundant M@C60 fraction of metallofullerenes, obtaining by a non-chromatographic procedure 13, 14.

The Gd3N@C80 fullerene exhibiting the highest relaxivity has seen wider use because of the presence of three Gd3+ ions per molecule. Functionalized trimetallic nitride endohedral metallofullerene species Gd3N@C80 [DiPEG5000(OH)x] has been used to perform high water hydrogen MR imaging relaxivity (R1) when used at a lower concentration, with an equivalent efficiency compared with commercial agents. After it was directly infused into a tumor bearing brain, improved tumor delineation was possible in comparison with the intravenously injected conventional Gd3+ chelate 15. Zhang et al. also compared the 1H MRI relaxivities of Gd3N@C80 fullerenes with PEG of four different molecular weights (350-5000 Da), and found that 350/750 Da PEG derivatives exhibited the highest relaxivity compared to longer chain length ones, 5000 Da PEG derivatives 16. At nearly the same time, Gd3N@C80(OH)26(CH2CH2COOM)16 (M=Na or H) was readily synthesized by Shu et al. with high yield. The longitudinal and transverse relaxivities R1 and R2 were 50 times larger than those of Gd3+ poly(aminocarboxylate) complexes, such as commercial Omniscan and Magnevist. Furthermore, the new derivatives were more compact, diffused more slowly and lingered for a longer time than commercial agents and Gd3N@C80[PEG5000(OH)x] 17. These results gave greater impetus to the development of metallofullerenes as MRI contrast agents.

In the previously mentioned studies, most imaging was obtained by directly administering fullerenes into tumor tissue, therefore tumor-targeting is certainly a subsequent challenge for tumor imaging. Antibodies of the green fluorescent protein (anti-GFP) were conjugated to endohedral gadofulleride, Gd@C82O6(OH)16-(NHCH2CH2COOH)8, as a model for “tumor-targeted” imaging agents, and could be detected by using a fluorescent GFP probe. Results revealed that the antibody activity was well preserved after the conjugation, and the MRI activity was higher than that of unconjugated gadofulleride 18. Recently, a more encouraging result was obtained. A carboxyl functionalized metallofullerene Gd3N@C80(OH)26(CH2CH2COOH)16 was successfully conjugated to an interleukin-13 (IL-13) peptide, the receptor of which was expressed on glioblastoma multiforme cells. This preliminary finding exhibited the potential of functionalized gadolinium metallofullerenes for tumor targeting and imaging 19.

Photoacoustic imaging

Recently, an attractive new therapeutic concept called 'acoustic-explosion' was suggested for some functionalized fullerenes, polyhydroxy fullerenes (PHF; C60(OH)xOyNaz) and carboxy fullerenes (CF; C60(C(COOH)2)3). Concomitant with the exposure to low-intensity (< 102 W cm-2) continuous-wave laser irradiation in the presence or absence of oxygen, sustained glowing of functionalized fullerenes was observed. When this process was performed in cancer cells, a faint pop was heard following irradiation, indicating the photoacoustic properties of functionalized fullerenes. To investigate their potential for cancer imaging, researchers then used a mechanical scanning photoacoustic system with a single transducer to collect the acoustic signal, and embedded it in a phantom together with functionalized fullerenes. After laser irradiation in a water tank, high contrast photoacoustic images of functionalized fullerenes were obtained. Similarly, the photoacoustic imaging experiment yielded promising results in tumor-bearing nude mice. Excellent contrast between tumor and non-tumor regions was shown after intratumoral injection of PHF, and the laser energy used was only one-third of the maximum permissible exposure level of 29.5 mJ cm−2 for a 785 nm pulsed laser as established by the American National Standards Institute 20, 21. This new finding suggests potential applications of functionalized fullerenes for obtaining photoacoustic images (Figure ).

Functionalized fullerenes for tumor therapy

Photodynamic therapy

Photodynamic therapy (PDT) is a non-invasive treatment for various types of tumor. The combination of a photosensitizing agent and focused irradiation is used to elicit specific, controlled production of reactive oxygen species in a localized area, leading to cell destruction through several pathways. By using highly focused light irradiation, photodynamic therapy has the potential to act specifically at the desired site of action with low levels of collateral damage to healthy cells 22. Fullerenes can efficiently generate reactive oxygen species when exposed to visible light, which means it may be an effective cytotoxic agent. The anatomical characteristics of tumor, such as hyperpermeable vasculature and an immature lymph system, make large-sized substances accumulate and remain longer in tumor tissues than in normal tissues. After the ideal molecular size, water-solubility, and tumor targeting were developed through various modifications, functionalized fullerenes are now available for cancer PDT both in tumor cells and tumor-bearing mice 23-25. Many researches have been carried out to demonstrate this potential application of functionalized fullerenes.

When C60 was conjugated with polyethylene glycol (PEG), pullulan, etc., strong tumor suppression was shown after the conjugate was injected intravenously into tumor-bearing mice coupled with light irradiation 26, 27. To confirm tumor uptake of C60(OH)x, 125I-labeled fullerenol, 125I-C60(OH)x were administered to five tumor-bearing mice. The accumulation ratios of 125I-C60(OH)x in tumor compared to normal muscle tissue (T/N) and blood (T/B) at different time points revealed that C60(OH)x could be accumulated in several tumors readily and persistently, especially in mouse H22 hepatocarcinoma, suggesting that it could be used as a good photosensitizer in photodynamic tumor therapy 28. To elevate the PDT effect by controlling the treatment time, Gd3+-chelated C60-PEG (C60-PEG-Gd) was obtained from mixing gadolinium acetate solution with C60-PEG-DTPA, which was prepared by introducing diethylenetriaminepentaacetic acid (DTPA) to the terminal group of C60-PEG. The MRI activity was introduced into the C60-PEG of PDT photosensitizers by the chelate incorporation of Gd3+ ions and provided a non-invasive way to detect the time course of C60-PEG accumulating in the tumor tissue. There was good agreement between the time profile of the PDT effect and the detection of a positive MRI signal. These results indicated that C60 could be a potent photosensitizing agent for photodynamic tumor therapy after proper modifications 29.

Two oxidative mechanisms are considered to be mainly implicated in the photodamage of cells. One is free radicals and the other is singlet molecular oxygen (1O2), generated through electron transfer and energy transfer, respectively. These two mechanisms may be determined by some important factors. For example, C60 induced DNA cleavage occurs mainly through reduced active oxygen radicals such as superoxide anion radical (O2—·) and hydroxyl radical (·OH) under physiological conditions, while 1O2 was generated effectively in nonpolar solvents 30. Besides, aggregation is a well-accepted factor that could deactivate the excited electronic states of photosensitizers and cause further loss of photoreactivity. Zhao et al. found that γ-cyclodextrin bicapped pristine C60 [(γ-CyD)2/C60] was in a monomeric state in water whereas C60(OH)24 was aggregated. It could be used to explain the phototoxicity induced by (γ-CyD)2/C60 toward human keratinocytes. The toxicity was mainly mediated by singlet oxygen and much higher than its water-soluble derivative C60(OH)24 31, 32. Compared to Type 2 photochemistry mechanism, Type 1 is more depend on the surrounding oxygen concentration; thus, some functionalized fullerenes acting through the Type 1 pathway may be relatively more effective in hypoxic tumors. C60-porphyrin could induce HepG2 human larynx-carcinoma cell death through either a 1O2-mediated photoreaction process or a free radical mechanism under anaerobic conditions 33. N-methylpyrrolidinium-fullerene (BB4) was found to be a powerful photosensitizer with the ability to kill cancer cells in vitro and in a mouse model of disseminated peritoneal carcinomatosis after illumination with white light. BB4 can induce apoptosis by producing Type 1 ROS, rather than singlet oxygen, i.e. a Type 2 mechanism 34, 35.

Photothermal treatment

Recently, certain functionalized fullerenes, especially polyhydroxy fullerenes (PHF) and carboxy fullerenes (CF) were found to be heated to their ignition temperature by exposure to low-intensity (< 102 W cm-2) continuous-wave laser irradiation, which was likely because of distortion of the symmetrical cage structure. This heating property would be extremely advantageous for cancer therapy, when a negatively charged PHF coating on silica nanoparticles functionalized with positively charged amine groups was dosed to A549 cells and localized destruction of cells was induced by irradiation with a near-infrared laser 21.

Moreover, when tumors were exposed to near-infrared light after injected with PHF nanoparticles, they decreased in cross-sectional area by an average of 32% within 2 hours of treatment, with only a blister visible 20 hours post-treatment. In addition, a concomitant faint audible sound was heard accompanied by bubble formation and bursting of the irradiated cells after exposure to a 785 nm laser. Thus, the rapid tumor shrinkage may be explained by both photothermal ablation and “acoustic-explosion” mechanisms (Figure ) 20.

Therapeutic Nuclear Medicine

While the potential of metallofullerenes on MRI was developed vigorously, research on another application of functionalized fullerenes to therapeutic nuclear medicine was also performed. Based on a similar principle, metallic radionuclides encapsulated in fullerenes can be protected in the empty carbon cage stably, eliminating any undesired toxicity induced by the leakage and catabolism of administered radionuclides.

The availability of radio-fullerenes in therapeutic nuclear medicine was then proven by distribution and stability evaluations. Diener et al. prepared the α-emitting 212Pb@C60 malonic acids for the first time and investigated the bio-distribution of the untargeted water-soluble radio-fullerene in mice and its stability during β--decay of 212Pb to 212Bi. It was shown that 212Pb@C60 malonate prevented the 212Pb from accumulation in bone, attenuated the myelotoxicity of 212Pb, and exhibited a rather slow clearance. This radio-fullerene also appeared stable in solution (about 36% of the 212Bi released) 36. The above results suggested that water-soluble fullerenes might be suitable as cancer radiopharmaceuticals.

Effective targeted delivery of radionuclides is the primary challenge in radio-immunotherapy (RIT). By conjugating the radio-fullerenes with an antibody that is specific for receptors expressed preferentially on the cancer cells, radioisotopes can be delivered to cancer cells. After encapsulation of the β-emitter 177Lu in a fullerene cage, an IL-13 peptide that targets an over-expressed receptor in glioblastoma multiforme tumors was successfully conjugated to the 177LuxLu(3-x)N@C80 37. However, the efficiency of these radio-fullerenes for tumor targeting RIT requires further investigation in the living body or tumor cells.

Chemotherapeutics

Besides surgery, chemotherapeutics is another principal method for tumor therapy. However, the high toxicity and easily developed drug resistance considerably confine the chemotherapeutic effect. In recent years, functionalized fullerenes have been found to have some good characteristics to aid with antitumor treatments, and some of them have exhibited the potential for tumor inhibition (Figure ) 9, 38, 39. In 2005, we found that gadolinium endohedral metallofullerenol (Gd@C82(OH)n nanoparticles) can efficiently inhibit the growth of murine H22 hepatoma without obviously adverse effects on important organs, having nearly no direct toxic effect to tumor cells 40, which is an advantage over conventional anti-tumor drugs. Zhu et al. 41 also tested the tumor-inhibitory effect of C60(OH)x on the same model. In the C60(OH)x-treated group, significant tumor inhibition rates and reduced damage to liver were shown, in accordance to the histological results of inhibited tumor infiltration. Additionally, the anti-metastatic activities of fullerenol C60(OH)20 was observed in cancer metastasis models 42. Moreover, some fullerenes could increase the chemo-sensitization of tumor cells to chemotherapeutic agents and result in antitumor drug-mediated cell death, especially in some drug-resistant cancer cells 43, 44. Thereafter, great attention was focused on the underlying mechanisms of the antitumor activity of fullerenes as follows:

1) Anti-angiogenesis

Angiogenesis is one of the most important factors for the progression of tumor, by supplying tumors with oxygen and nutrients 45. As tumors can generate their own blood vessels from the surrounding vasculature 46, 47, anti-angiogenesis treatments can inhibit growth and metastasis of various solid tumors 48.

Meng et al. investigated the anti-angiogenic activity of Gd@C82(OH)22 and found that the nanoparticle can simultaneously down-regulate more than 10 angiogenic factors, which was confirmed in vivo by decreasing tumor microvessel density and lowering the nutrient supply to tumor tissues. A similar result was also obtained in another anti-angiogenesis study of C60(OH)20 (Figure ) 42, 49.

2) Modulation of oxidative stress

ROS such as superoxide radical anion, hydrogen peroxide, singlet oxygen, and hydroxyl radical, which can cause oxidative stress, are considered to be an important part in the mechanism of carcinogenesis. ROS can induce lipid peroxidation, protein fragmentation, DNA damage, damage to tumor-suppressor genes and enhance the expression of proto-oncogenes. It has been reported that various carcinoma cells synthesize ROS at an elevated rate in vitro, and many tumors are under persistent oxidative stress in vivo 42. As water-soluble fullerenes showed high inhibitory activity on many kinds of tumor growth in mice, and it is well established that fullerenes and their derivatives possess a unique capacity for scavenging ROS 50, the relationship between the two functions of the nanoparticles was investigated.

The ROS scavenging capability of [Gd@C82(OH)22]n was verified by electron spin resonance (ESR) 51, as well as in tumor tissues of C60(OH)20 treated mice 42, which meant that the antitumor mechanism of fullerenes may be related to its capacity to modulate oxidative stress in tumor-bearing models. On the other hand, Injac et al. found that fullerenol C60(OH)24 had protective effects on the primary organs (heart, liver, lung and kidney) against chronic toxicity induced by doxorubicin, and thus may be useful as a potential organo-protector for anticancer therapy 52-55. These results may increase the feasibility of applying fullerenes to cancer chemotherapeutics.

3) Immunological activity

Our previous studies have demonstrated that water-soluble C60(OH)20 and Gd@C82(OH)22 nanoparticles have specific immunomodulatory effects on T cells and macrophages, and up-regulated the immune response in vivo, including the polarization of the cytokine balance towards Th1 (T-helper cell type 1) cytokines, which particularly increase TNF-α production, a very important factor helping to scavenge and kill tumor cells 9, 38. These results indicated effective immunological mechanisms of functionalized fullerenes (Figure ).

Zhang et al. found that Gd@C82(OH)22 could effectively activate dendritic cells (DCs) and macrophages, which are the most important antigen presenting cells (APC), as well as up-regulate the expression of co-stimulatory molecules and MHC molecules, thus enhancing the APC function of DCs and macrophages as illustrated by their capacity to activate allogeneic T cells. More evidence was also given to demonstrate the immune activity of Gd@C82(OH)22, which could increase the production of Th1 cytokines and promote macrophages secreting pro-inflammatory cytokines such as IL-6 and TNF-α 56, 57.

Moreover, there are few reports about the antigenicity of C60 derivatives. Chen et al. found that mice immunized with the fullerene-bovine thyroglobulin conjugates generated a population of fullerene-specific antibodies of the IgG isotype, including a subpopulation that was cross-reacted with a C70 fullerene 58. The specificity of the monoclonal antibody for the C60 fullerene was determined by competitive inhibition, and the anti-C60 antibodies were able to interact with single walled carbon nanotubes, which was imaged using atomic force microscopy 59, 60.

4) Overcoming tumor resistance to chemotherapeutic drugs

For cisplatin-resistant cancer (CP-r) cells, Gd@C82(OH)22 alone had no obvious inhibition on tumor growth, although it has been indicated that the metallofullerene could improve the sensitivity of CP-r tumors to cisplatin, as an effective inhibition of CP-r tumors growth was observed following cisplatin plus nanoparticle treatment in vitro and in vivo. Transferrin-labeled experiments have shown that less intracellular transferrin is distributed in CP-r cells than in CP-s cells, indicating a possible defective endocytosis of the CP-r cells. Since nanoparticles have been used as drugs or gene carriers for cancer treatment, it was posited that [Gd@C82(OH)22]n nanoparticles reverse tumor resistance by enhancing the endocytosis of cisplatin via nanoparticle-mediated penetration through the plasma membrane of the CP-r cells, and this hypothesis was confirmed by increased cisplatin adducts in NP and CP-treated CP-r cells measured by ICP-MS. Restoring defective endocytosis and other related possible mechanisms for overcoming tumor resistance to cisplatin by using fullerenes could lead to new chemotherapies for cancer 43.

5) Autophagy-mediated chemo-sensitization

The main therapeutic goal of cancer treatment is to trigger tumor-selective cell death. One of the cellular pathways receiving much attention in recent years is autophagy, a highly-regulated intracellular process for the degradation of long-lived proteins and damaged organelles. Autophagy can be either a cell death pathway or a cell survival pathway depending on many factors such as the degree of cell damage and intactness of apoptotic pathways 61. Undoubtedly, promoting autophagy-mediated cell apoptosis is of most concern for tumor therapy.

Zhang et al. have performed comprehensive work on the modulation of autophagy in cancer cells by fullerene C60 nanocrystals. They found that HeLa cells treated with nano-C60 exhibited autophagic features, such as LC3 puncta formation, and many more autophagic vacuoles including autophagosomes and autolysosomes. Nano-C60 at non-cytotoxic concentrations sensitized chemotherapeutic killing of cancer cells in a photo-enhanced and ROS-dependent pathway, which was further verified via GFP-LC3 dot formation that was effectively reduced by free radical scavengers in the dark treatments 44. A much greater potential in the induction of autophagy by another C60 derivative, C60(Nd), was also reported 62. All above results prompt that fullerenes might be available to be an efficient chemo-sensitizer in cancer therapy.

Bio-distribution and Toxicity

Although the diverse potential applications of fullerene and its derivatives have been exploited in cancer diagnosis and therapy, it is necessary to consider their biocompatibility and toxicity for organisms prior to their widespread utilization and production. Since fullerene derivatives with different modifications have distinct biological behaviors, understanding these will help in the safe development of fullerene-based drugs 63, 64.

Bio-distribution and excretion

The dynamic distribution of fullerenes within the body is influenced by several factors, including the particle size, various modifications, and administration pathways. Studying these factors can help to identify potential target tissues of fullerene and assess the toxicity at particular target sites.

Unlike oral administration, whereby 14C-labeled C60 is not efficiently absorbed and excreted primarily in the feces, C60 injected intravenously was cleared from the blood circulation rapidly, accumulated mainly within the liver and remained there for a long time, and then was cleared slowly from the liver (nearly all was eliminated 13 days after injection). All these results suggest that the liver is a potential target for fullerene accumulation and toxicity contributes to the metabolism of C60 (C60 metabolites were identified within the liver) and facilitates the removal of fullerenes from the body within the feces 65-67. By comparison, the water-soluble C60 had a wider tissue distribution, which might be attributed to its more hydrophilic water-soluble character. 99mTc-C60(OH)x injected into mice through tail veins was quickly distributed to all organs except for tissues with limited blood flow, such as muscle. It remained throughout the 48 h of the study, particularly in the kidneys, bone, spleen and liver, and displayed a slow clearance rate except for bone, which showed slightly increasing accumulation within 24 h 68. This phenomenon was further demonstrated by studying the distribution of 125I-C60(OH)x, which was rapidly distributed to all organs (mainly in the kidneys, liver, bone, spleen and stomach) through the blood circulation and especially exhibited a rapid and high uptake in kidneys, followed by a fast excretion into urine. Likewise, 125I-C60(OH)x was not found in the brain, suggesting it could not penetrate the blood-brain barrier (BBB), which was different from the C60 modified with dipolar trimethylenemethane 28.

Similarly, Cagle et al. 69 found that 166Ho@C82(OH)y was distributed throughout the entire body, except for the brain and fat tissues, as early as 1 h after the tail-vein injection, which was different from the finding with metal chelate (Na2[166Ho(DTPA)(H2O)]). Only the concentrations of 166Ho in liver and bone increased from 1 to 4 h, especially in bone, which subsequently displayed a slight increase in concentration over 48 h (Figure ). These results were consistent with the observations for the bio-distribution of [Gd@C82(OH)22]n particles. Unlike GdCl3, [Gd@C82(OH)22]n was delivered to almost all observed tissues, but accumulated mostly in bone, suggesting that the fullerene cage could not be destroyed in organisms and that the internal Gd3+ was well protected in the cage. The concentration of Gd in the brain was close to the baseline, suggesting that the [Gd@C82(OH)22]n nanoparticles could not pass through the BBB either (Figure ) 70. The similar results for the bio-distributions of C60(OH)x and C82(OH)y showed that the biological properties would not be expected to change by incorporation of a metal atom within a fullerene cage. Thus, distribution and biocompatibility of fullerenes were usually assessed through ip and iv injections, and chemical modification to the fullerene cages may play an important role in determining their distribution and biocompatibility.

Toxicity evaluation

To understand the toxicity of C60, it is usually necessary to investigate the lethality, gross or microscopic lesions, histopathological change, etc. However, other biological responses, such as stimulation of the immune system, regulation of oxidative stress, and changes on the molecular level induced by fullerenes, should generally be considered as well.

Following the inhalation pathway, C60 and its derivates can be partly deposited in the lungs and internalized by alveolar macrophages; a pulmonary inflammatory response was elicited for a high exposure dose of fullerenols (10 mg/kg) and fullerenols at low concentrations (1 mg/kg) exhibited an anti-inflammatory property. Although slight changes in gene expression level were observed, no obviously toxicity was reported 71, 72. Only when water-soluble polyalkylsulfonated C60 was administrated at extremely high dose (LD50=600 mg/kg) in an acute toxicity study or at a moderately high dose (up to 60 mg/kg) via ip injection, observable pathological or lethal toxicity was induced. However, in subsequent study of oral exposure, fullerenes were considered to be nontoxic at a dose of 50 mg/kg, which was close to that of ip administration 73. Even when the dose was increased to 2,000 mg/kg, fullerite (a mixture of C60 and C70) has not shown to cause any acute toxicity. The possible reason is that fullerenes are not effectively absorbed through the alimentary tract 74. Toxicity assessment of fullerenes should therefore be considered comprehensively, including the exposure routes and the delivered dose, etc.

After fullerenes enter into the body, they may be internalized by different types of cells, and induce various changes inside the cells, such as viability, proliferation, inflammation responses, and oxidative responses. Therefore, the cytotoxicity assessment of fullerenes has been one of the most important aspects to consider when assessing the toxicity of fullerenes 75. Many related studies have been performed on various cell lines. C60 and quaternary ammonium derivatized C60 inhibited keratinocytes proliferation at a concentration of 2 μM and over a period of 8 days, which is relatively long 66. In both human skin (HDF) and liver carcinoma (HepG2) cells, water-soluble fullerene species are less cytotoxic than pristine C60, while C60(OH)24 showed no cytotoxicity up to its limits of solubility, suggesting the functional groups on the surface of a fullerene molecule can dramatically decrease the toxicity. However, C60(OH)24 was found to induce cytotoxic morphological changes within the HUVEC cell line in a dose-dependent way, and only maximal doses of fullerenes caused cytotoxic injury and/or death and inhibited cell growth 76. In nearly every fullerene-related anti-tumor study, fullerenes were not found to have a direct influence on tumor cell viability 9, 38, 40, 42. The diverse toxicity results of fullerenes reveal that the sensitivity of various tissues or cells to the nanoparticles can be different. Therefore, thorough toxicity evaluation of fullerenes before their clinical application is necessary.

Perspectives

Due to their attractive physical, chemical, and biological properties and the availability of various methods for surface modification, fullerenes and their derivatives have been studied for their numerous biomedical applications, not only in tumor diagnosis and therapy, but also as an anti-HIV agent 77, 78, antimicrobial photosensitizer 79, 80, antioxidants 52, 81, 82, and gene vector 83-85. Some properties involved in these applications might create new insights into fullerenes as anti-tumor therapeutics, for example, in gene therapy. However, since the underlying mechanisms related to their apparent activities are not yet clear, further research towards a better understanding is urgently needed to investigate the biocompatibility and safe application of functionalized fullerenes as diagnostic tools and therapeutic agents.

Table 1

Application of various functionalized fullerenes to tumor theranostics.

Applications		Types of functionalized fullerenes	Predominant characteristics	References
Cancer diagnosis	Magnetic resonance imaging (MRI)	Gd@C82(OH)40	higher signal enhancement at a dose of 1/20 of commercial Gd-DTPA	10
		Gd@C82O2(OH)16(C(PO3Et2)2)10	high affinity to bone	11
		Gd@C60[C(COOH)2]10	excellent cellular uptake efficiency without any adjunctive agent, obtained by a non-chromatographic procedure with more abundant M@C60 fraction	13, 14
		Gd3N@C80[DiPEG(OH)x]	the highest relaxivity	15, 16
		Gd3N@C80(OH)∼26(CH2CH2COOM)∼16(M=Na or H)	readily synthesized with high yield, further qualified diffusion time	17
		Gd@C82O6(OH)16-(NHCH2CH2COOH)8Gd3N@C80(OH)26(CH2CH2COOH)16	conjugated to an protein/peptide for “tumor targeted” imaging	18, 19
	Photoacoustic imaging(&Photothermal treatment)	C60(OH)xOyNaz	“acoustic-explosion” after exposure to laser irradiation	20, 21
Cancer therapy	Photodynamic therapy	C60 conjugated with PEG,/pullulan	strong tumor suppression coupled with light irradiation	26, 27
		125I-C60(OH)x	accumulated in tumors readily and persistently	28
		C60-PEG-Gd	a good agreement between the time profile of the PDT effect and the detection of positive MRI signal	29
		(γ-CyD)2/C60	singlet oxygen -mediated phototoxicity	31, 32
		C60-porphyrin	induce carcinoma cell death through either a 1O2-mediated photoreaction process or a free radical mechanism	33
		N-methylpyrrolidinium-fullerene (BB4)	induce apoptosis by producing Type 1 ROS	34, 35
	Radiotherapy	212Pb@C60 malonic acids	stable during β--decay of 212Pb to 212Bi, attenuates the myelotoxicity of 212Pb	36
		177LuxLu(3-x) N@C80	conjugated to IL-13 for RIT	37
	Chemotherapeutics	Gd@C82(OH)22	efficiently inhibit the growth of murine H22 hepatoma with low toxicity, reverse tumor resistance by enhancing the endocytosis of cisplatin via nanoparticle-mediated penetration	40, 43
		C60(OH)x	significant tumor inhibition, anti-metastatic activity	41, 42
		C60, C60(Nd)	induce autophagy-mediated chemosensitization	44, 62