Structure of C60 Fullerene Nanoparticles in Aqueous Colloid Solution
Knowledge of the C60 fullerene aggregation parameters in aqueous solution is important for estimation of nanostructure bioactivity and its potential for biomedical application [18, 24, 25]. An AFM investigation was performed to characterize the aggregation state of C60 fullerene in aqueous solution.
The AFM image (Fig. 1) demonstrates that, along with the individual C60 fullerenes, their bulk aggregates (clusters) with a diameter of up to 50 nm were present on the mica substrate. These results are in a good agreement with the data from laser correlation spectroscopy of C60FAS, which confirm that the average hydrodynamic diameter of nanoparticles is 50 nm and no further agglomeration is observed . More detailed analysis of the structural composition and physico-chemical properties of C60FAS has been previously accomplished [21, 23] by means of chemical analysis, ultraviolet-visible spectroscopy (UV/VIS) and Fourier transform infrared spectroscopy (FTIR), scanning tunneling microscopy, dynamic light scattering, and small-angle neutron scattering techniques. In general, the properties of the C60FAS used in this experiment correspond to those in the published literature data.
Tumor Growth-Inhibitory Effects
In traditional animal tumor models, Dox is typically introduced at concentrations of up to 100 mg/kg; however, high lethality complicates prolonged monitoring of Dox effects. Injection of the chemotherapeutic agent in lower doses is more effective than a single injection at an equivalent dose .
In our experiments, we used a model of multiple ip injections for a course of Dox treatment with a total dose of 2.5 mg/kg (corresponding to a minimal therapeutic dose), and in combination with C60 fullerene as a potential antitumor agent and protector against Dox-induced toxicity. The total dose of C60 fullerene (25 mg/kg) was non-toxic as expected, since the median lethal dose (LD50) for pristine C60 fullerene ip injected in a water solution to mice was found to be approximately 600 mg/kg .
The kinetics of average tumor volume extension that manifests the tumor growth was measured in three experimental groups of animals from the 7th to 22nd day after tumor inoculation and compared with that of the control cancer group. The tumor growth inhibition curves for the various groups are shown in Fig. 2. The tumors grew the quickest in the control group. Tumor inhibitory effect in the C60 fullerene-treated group (22.5 %) was comparable with that in the Dox-treated group (24.2 %). When C60 fullerene was administered before Dox (C60 + Dox group), a stronger inhibitory effect was observed (36.2 %). Thus, these data indicate that treatment with C60 + Dox more effectively inhibits tumor growth than does treatment with Dox or C60 fullerene separately.
Quantitative Indexes of Antitumor Effect
Data on the lifespan of the control and treated groups are presented in Table 1. The last animal in the control cancer group died on the 30th day of the experiment, whereas the lifespan of animals in experimental groups was found to be increased. The last animal in the Dox group died on the 38th day, and those in the C60 fullerene and C60 + Dox groups died on the 40th day.
Table 2 shows the quantitative indexes of animal life prolongation (k
p) and metastasis inhibition (k
m) as the measure of the total effectiveness of the applied regimes of antitumor treatment.
LLC cells are highly invasive and quickly spread into lung tissue and form multiple metastases. Dox treatment distinctly inhibited tumor dissemination into the lung of tumor-bearing animals (k
m ~ 23 %) but exerted only a slight effect on life prolongation (k
p ~ 16 %). In the C60 fullerene-treated group, the metastasis inhibition index was less than that in the Dox-treated group (k
m ~ 15 %), but the life prolongation was greater (k
p ~ 24 %). Both the anti-metastatic effect (k
m ~ 35 %) and the life prolongation (k
p ~ 32 %) were found to be maximal in the C60 + Dox group.
Cytomorphological Changes in Tumor Tissue
Cytomorphological study of the tumor that developed subcutaneously in mice in the cancer control group (Fig. 3a) showed that it consisted of polymorphic cells with a nucleus, large nucleolus, condensed chromatin, and light areas of tumor karyoplasm. The high content of mitotic cells testifies to the proliferative potential of tumor cells. In the C60 fullerene-treated group, the tumor mitotic index did not change, but the apoptotic index was found to be higher than in the control cancer group (Figs. 3b and 4). Dox treatment was followed by a reduction in the number of mitotic cells and an increase in the number of apoptotic cells, i.e. by inhibition of proliferative potential and induction of tumor cell death (Figs. 3c, 4). When the tumor-bearing animals were exposed to treatment with C60 + Dox, the number of apoptotic cells substantially increased and zones containing apoptotic, necrotic cells and infiltrating macrophages were detected (Figs. 3d and 4).
Activity of Antioxidant Enzymes in Liver and Heart
Dox chemotherapy is known to be accompanied by the generation of reactive oxygen species (ROS) and immediate oxidative damage of cells with high oxidative metabolism and activity of mitochondrial respiratory chain, particularly cardiac myocytes and hepatocytes. SOD and GP are the predominant antioxidant enzymes. The first step of antioxidant defense is the dismutation of superoxide anion into hydrogen peroxide by inducible SOD; the second is conversion of hydrogen peroxide to water by GP. The activity of SOD and GP was estimated in the liver and heart of animals in the cancer control, Dox-, and C60 + Dox-treated groups. No reliable difference in the level of heart and liver antioxidant enzymes in the C60 fullerene group as compared with the cancer control group was observed.
Prolonged Dox administration resulted in a decrease of SOD as well as of GP activities in both mice liver and mice heart (Fig. 5). Since activity of both enzymes is known to undergo feedback inhibition by excessive levels of peroxides and lipoperoxides, it could be that Dox-dependent oxidative stress diminishes ROS-scavenging activity of SOD and GP in the liver and heart of tumor-bearing animals.
No inhibition of antioxidant enzyme activity was detected in the C60 + Dox-treated group; the level became comparable with that in the control group (SOD activity in liver and heart and GP activity in heart) or even exceeded that in the control group (GP in liver) (Fig. 5). This favorable phenomenon is likely to be associated with the ability of C60 fullerene to function as a ROS scavenger or SOD mimetic  and to decrease the production of ROS to the optimal level for antioxidant enzyme activation. Up-regulation of SOD has previously been shown to enhance cell survival in the presence of Dox through its role as a free radical scavenger . In addition, overexpression of cytosolic and mitochondrial GP appeared to protect mice hearts against Dox-induced cardiotoxicity and to prevent impairment of mitochondrial respiration and inhibition of complex I activity .
This study has shown that the treatment of tumor-bearing mice with ip injection of C60 + Dox from the 7th day after LLC cell inoculation resulted in tumor growth slowdown, metastasis inhibition, and lifespan prolongation. These indexes were also increased after Dox or C60 fullerene were administered as single agents, but the antitumor effect was most pronounced in the C60 + Dox group. The histological analysis of tumor tissue in the C60 + Dox-treated group confirmed the inhibition of proliferating tumor cells, induction of apoptotic and necrotic cell death, and macrophage infiltration.
In several previous in vitro studies, internalization of pristine C60 fullerene into cancer cells was demonstrated, but no cytotoxic effects were observed. When human lung carcinoma A549 cells were exposed to pristine C60 fullerene dispersion (particle size 100–200 nm), internalized C60 fullerene aggregates were detected in the cytoplasm and lysosomes. Neither apoptosis nor necrosis was induced, while cell proliferation was inhibited . Malignant breast epithelial cells were shown to take up pristine C60 fullerene from methanol C60 fullerene solution (particle size 106–342 nm), and the treatment of these cells with C60 fullerene up to 200 μg/ml did not alter the morphology, cytoskeleton organization, cell cycle dynamics, or cell proliferation .
Other studies on pristine C60 fullerene distribution in animal tissues after ip administration demonstrated nanoparticle uptake into the circulation, accumulation in the liver at 120 min post-exposure, and nearly complete elimination by day 13, with minimal accumulation in the spleen, lung, and muscle [6, 32].
Internalization of C60 fullerene in tumor tissue has also been demonstrated with in vivo models and different water-soluble C60 fullerene derivatives. In a mouse model of liver cancer, malonodiserinolamide-derivatized C60 fullerene nanoparticles were detected permeating through the altered vasculature of the tumor, evading the reticulo-endothelial system . Gd@C82(OH)22 nanoparticles administered ip at a dose level of approximately 10−7 mol/kg were shown to exhibit high antineoplastic activity against murine H22 hepatocarcinoma, but only 0.05 % of the exposed dose reached the tumor tissue . It was also demonstrated that fullerol C60(OH)x nanoparticles were mainly taken up by the mononuclear phagocyte system, and only 0.2 % were accumulated in the murine H22 hepatocarcinoma within 24 h due to enhanced permeability and retention effects . Nevertheless, C60(OH)x in doses of 0.2 and 1 mg/kg was found to exhibit significant tumor-inhibitory activity .
Considering that no marked accumulation of C60 fullerene was detected in tumors and no direct toxic effect on tumor cells was observed, it is assumed that antitumor efficiency of C60 fullerene derivatives is not due to the direct killing of tumor cells but is the result of tumor microenvironment regulation and/or immune response activation [12, 36–38]. On the other hand, our finding of C60 fullerene-dependent enhancement of Dox antitumor effect does not exclude the possibility that C60 fullerene nanoparticles promote Dox accumulation in tumor cells by activating drug endocytosis, as it was shown for Gd@C82(OH)22 nanoparticles that may restore defective endocytosis of cisplatin by cancer cells .
The most severe side effect of Dox treatment is free radical-mediated damage to liver and heart tissue, which are very sensitive to free radical damage because of their high oxidative metabolism. In this study, we have demonstrated a beneficial effect of pristine C60 fullerene in a low-dose regime (5 mg/kg) on heart and liver antioxidant defense against Dox treatment in tumor-bearing mice. The greater protective effect of lower doses of fullerols (25 mg/kg) in contrast to higher doses (50 and 100 mg/kg) against Dox-associated toxicity was confirmed for rat hearts and livers with colorectal cancer [3, 40]. This observation is likely to be associated with the fact that the higher doses were less well absorbed from the peritoneal cavity.
In summary, we can conclude that pristine C60 fullerene exhibits health effects and antitumor activity in combined treatment with Dox. These results need to be investigated further in a number of tumor models.