The C 60 ( FeCp 2 ) 2-Based Cell Proliferation Accelerator

1 Laboratory for SuperconductingMaterials Physics, State Scientific and ProductionAssociation “Scientific-PracticalMaterials Research Center of the NAS of Belarus”, P. Brovka Street 19, 220072 Minsk, Belarus 2 Department of New Materials, Lykov Institute of the Heat and Mass Transfer of the NAS of Belarus, P. Brovka Street 15, 220072 Minsk, Belarus 3 Central Scientific Research Laboratory of the Belarus State Medical University, Dzerjinski Avenue, 83, 220116 Minsk, Belarus 4 Institute of Physiology of the NAS of Belarus, Academicheskaya Street 28, 220072 Minsk, Belarus 5 School of Chemistry, Manchester University, Oxford Road, Manchester M13 9PL, UK


Introduction
The last two decades studies have showed that fullerene C 60 derivatives exhibit a great potential in many fields of biology and medicine [1].These are UV and radioprotection [2], specific DNA cleavage [3], antiviral, antioxidant, and antiamyloid activities [1,4], allergic response [5] and angiogenesis [6] inhibitions, immune stimulating and antitumor effects [7,8], enhancing effect on neurite outgrowth [9], and gene delivery [10].Fiorito and coauthors showed that Cfullerenes, when highly purified, do not stimulate the release of NO by murine macrophage cells in culture, their uptake by human macrophage cells is very low, and they possess a very low toxicity against human macrophage cells [11].Watersoluble fullerene derivatives protect human keratinocytes from UV-induced cell injuries together with the decreases in intracellular ROS generation and DNA damages [12] and suppress intracellular lipid accumulation [13].Ferrocene derivatives in particular ferrocenium salts demonstrate antitumor activity [14,15].Authors [16] proposed possible mechanisms of action of these compounds on tumors, which can be assumed as one-electron oxidation of the ferrocene nuclei to the ferrocenium salts Fc + X − .These salts have relatively low reduction potentials fit for biochemical process to take place [17].
Many medical applications need that the drug materials possess ferromagnetic properties at room temperature.This allows managing the drug moving by magnetic field.ESR measurements on C 60 (FeCp 2 ) 2 over a wide temperature range (4-290 K) indicate the formation of a low concentration of C 60 − ions and thus the presence of weak charge transfer interactions of C 60 with ferrocene and the possible occurrence of at least a small amount of ferrocenium cations [18].The C 60 − ESR spectra show appreciable anisotropy at  < 40 K, reflecting the freezing of the molecular rotation and the static distortion of the fullerene local symmetry similar to other fulleride salts.Moreover, the temperature variation of the C 60 − ESR spectra at low temperatures ( < 10 K) suggests the presence of antiferromagnetic spin-spin interactions.A major contribution of iron ions and ferromagnetic particles is identified at  < 100 K that may account for the dc magnetic response of the compound.These allow us to believe that ferrocene fullerides will have a good perspective for biomedical applications.
In this work, we studied structural and magnetic proprieties of the fulleride C 60 (FeCp 2 ) 2 .The influence of fulleride particles on the cell proliferative activity was also investigated.

Experimental
Single crystalline specimens of C 60 (FeCp 2 ) 2 were grown from the solution of a stoichiometric mixture of C 60 and FeCp 2 in benzene by slow evaporation at 300 K as described in [19].
In order to study the influence of the fulleride presence on cell proliferation, we chose the cell line rin-mf5 (cell derivatives of the human insular tissue).These cells possess the large receptor field and high sensitivity against an attack of no specified cell toxins.These properties make them suitable targets for biotropic agents.Under the experimental conditions studied, substances were introduced directly into culture medium of the cells rin-mf5, grown in the RPMI 1640 medium.The results were examined in comparison with the cell amount grown in the same conditions without any additive.Data simulation was carried out with the "StatOlympus3X" software.
Magnetic measurements of powdered crystals were performed on the magnetometer Quantum Design MPMS XL 7T.X-ray analysis was carried out on the DRON-3 diffractometer.

Results and Discussion
As grown single crystals of C 60 (FeCp 2 ) 2 have pine-like shape (see Figure 1) and a brown color, no impurities have been detected by X-ray analysis.C 60 (FeCp 2 ) 2 was synthesized first by Crane with coauthors [19].This matter has triclinic structure with space group of Pī.Our X-ray analysis data is in agreement with results of [19].
Large crystals can be used as a substrate for adhesion and growth of cells (see Figure 2(b)).At the same time, the crystals with size of more the 10 m do not have reliable influence on the proliferative activity of the cells in monolayer culture (Figure 2(c)).After 24 hours of incubation, the cell amount in the experimental samples ( = 6) was larger (but less than 40%) than that observed in control samples.The most intelligible effect of the proliferative activity was found in presence of the C 60 (FeCp 2 ) 2 nanoparticles.The proliferative activity of the rin-mf5 cells increases (53% versus control shown at Figure 2(a)) in presence of the C 60 (FeCp 2 ) 2 nanosized particles (500 particles/cm 2 ).Moreover, it was registered that at the places where the fulleride particles are accumulated, the cell culture became multilayered and secreted basophile matrix (see Figure 2(d)).
To our opinion, it can be related to the particle size of the fulleride nanocrystals C 60 (FeCp 2 ) 2 , which probably contain the Fc + C 60 − salt.The possible mechanism of the proliferation acceleration consists in nonspecific endocytosis of fulleride nanoparticles by rin-mf5 cells.Due to relatively low reduction potentials Fc + cation that is suitable for intracellular biochemical process to take place, the C 60 (FeCp 2 ) 2 nanoparticle can be a catalyst of the intracellular regulatory systems and accelerate the rin-mf5 proliferation.
Magnetic measurements were performed by heating from 2 K up to room temperature under the magnetic field of 1 T. The results do not confirm the existence any ferromagnetic or antiferromagnetic ordering of C 60 (FeCp 2 ) 2 as described earlier in the investigated temperature interval.As shown in Figure 3, C 60 (FeCp 2 ) 2 is paramagnetic at liquid helium temperature.During the heating, it was transforming to the diamagnetic state near 60 K and was not changing the magnetic state up to room temperature.

Conclusion
C 60 (FeCp 2 ) 2 does not possess any ferromagnetic or antiferromagnetic ordering from 2 K up to room temperature.
The proliferative activity of the RINmF5 cells in presence of the C 60 (FeCp 2 ) 2 nanoparticles increases (53% versus control).It is proposed that the proliferation rate depends on the fulleride particle size.The possible mechanism of the proliferation acceleration consists in nonspecific endocytosis of fulleride nanoparticles by rin-mf5 cells.Due to relatively low reduction potentials Fc + cation that is suitable for intracellular biochemical process to take place, the C 60 (FeCp 2 ) 2 nanoparticle can be a catalyst of the intracellular regulatory systems and accelerate the rin-mf5 proliferation.

Figure 2 : 2 Figure 3 :
Figure 2: The rin-mf5 cell proliferation in presence of the C 60 (FeCp 2 ) 2 particles.(a) Cell proliferation (standard condition in monolayer).(b) Adhesion and growth of cells on the big crystal.(c) Influence of the crystals with size of nearly 5 m on the cell proliferation.(d) Cell proliferation in presence of the C 60 (FeCp 2 ) 2 nanoparticles.