The Development Of An Novel Nucleus Targeting Nanomaterial C60（OH）₂₄-NLC-E For The Radiation Protection

Background: It has been reported that free radicals such as H^., H₂, H₂O₂,H_aq-,OH., e_aq-play a key role in the radiation damage. The experiments of Templetetonand others had proved that90%of low LET rays-induced injury was caused by OH.which resulted in double strand break. DSB was one of the main reasons for cell deathas well as the most critical damage of DNA molecular structure and genetic integrity inwhich DSB can directly lead to chromosome aberration and genetic material losses. Sothe perfect free radical cleavage agents should be able to get into the cytoplasm,especially into the nucleus so as to clear up the free radicals surrounding the DNA andbiomembrane at the moment of ionizing radiation. Natural free radical cleavageagents,such as the enzymes superoxide dismutase, catalase, glutathione peroxidase andglutathione reductase, were unable to access to the nucleus and moreover did not affecton H_aq-,OH.and so on.Toxicological studies have showed that fullerene was a kind of low-toxiccompound. Its water-soluble and hydroxyl derivative fullerenol were good at removingthose free radicals and especially worked on the H_aq-and OH.with the reaction rateconstant up to （0.5-3.3）×1010M^-1S^-1. The fullerenol might pass through cell membranequickly and mainly distribute in the cytoplasm and cell organelles such as mitochondriabut cyteblast. Currently, the domestic and foreign relevant researches were mainly tocombine different chemical groups with fullerene to form new fullerene derivativeswhich can be applied widely. Nevertheless until now there is no report that fullerene orits derivatives had been made to act as radiation protection agents or free radicalcleavage working within the nucleus.Objective: To develop a special nanomaterial as a radiation protectant which candelivery the fullerenol into the cytoplasm and nucleus to remove the free radicalsefficiently. Methods: The fullerenols have been synthesized by the reaction of fullerene withNaOH solution in the presence of tetrabutylammonium hydroxide（TBAH）. Thesynthesized fullerol was characterizated with FT-IR, NMR and LC-MS. The newnanomaterial C60（OH）₂₄-NLC-E was prepared by high pressure homogenization-hothomogenization, which has high affinity with the nuclear receptor ER. The size anddistribution was detected by Laser High Performance Particles Sizer. The morphologywas observed by Scanning Electron Microscope. The ability of endocytosis wasobserved by laser confocal microscopy with Rhodamine-6G labeled C60（OH）₂₄-NLC-E.MTT assay was used to measure the toxical effect of nanoparticles on cultured V79cells.The radiation protection was determination by the cell cloning formation assay aswell as γ-H2AX foci countings and its luorescence intensity to represent DSB ofDNA.Results:（1） The new synthesized fullerenol sample displayed a strong and broadpeak at approximately3433cm-1for the hydroxyl groups on the fullerene cage by TheFT-IR spectrum. The1H-NMR spectrum of fullerol in deuterium DMSO showedevidently a proton peak centered at δ=3.34and two peaks centered at δ=2.50and δ=1.24,the latter was the proton peak for hydroxyl groups. From the mass spectrogram of thenew synthesized fullerenol, the major peak at m/z720was corresponding to themolecular mass of C60. In addition, a peak series from m/z720to1128with a spacingof m/z68was found, which suggest the addition of OH to the molecule. While anobserved peak at m/z1128could in theory correspond to a completely fullerol(C60（OH）₂₄).（2）The new propared nanomaterial C60（OH）₂₄-NLC-E presented a veryhomogeneous morphology with quite uniform particle size distribution and ratherspherical shape, and its particle size diameter was about300nm, these nanoparticles sizewere the same as that obtained by particle size analysis. The C60（OH）₂₄-NLC-E labeledwith rhodamine-6G was observed to enter into the cell quickly and obviously gatheredin the cytoplasm and nucleus. It was about15³0minutes to reach to saturationintracellular.（3）The cell survival rate was higher than95%when the end concentrationof C60（OH）₂₄-NLC-E was less than1.5μmol/L.（4）After irradiation by Colbat60-γ rayat dose from0to8Gy, the survival fraction of the cell treated with C60（OH）₂₄-NLC-E（1.27μmol/L） at0.5h before irradiation has increased significantly （p＜0.05） comparedwith the control cell. The D0and Dqof the treated cell were higher than the control cell significantly（p＜0.05）.（5）The γ-H2AX foci have been induced as early as30minutes after radiation. However, when C60（OH）₂₄-NLC-E was added30minute beforeIR, the counts of γ-H2AX foci reduced significantly （p＜0.05）.Conclusion: An novel nanomaterial C60（OH）₂₄-NLC-E has been developedsucessfully. The fullerenols can be deliveried into cell and concentrated in the targetedcytopasm and nucleus by C60（OH）₂₄-NLC-E. It can effectively scaveng free radicalsand directly protect DNA from the irradiation damage, which may be a potentialradiation protectant with advantage of high protecting efficiency and low toxicity.