FullerenolCytotoxic Conjugates for Cancer Chemotherapy Padmaparna Chaudhuri †,‡ Abhimanyu Paraskar, ,†,‡ Shivani Soni ,†,‡ Raghunath A. Mashelkar †,§ Shiladitya Sengupta †,‡, * † Laboratory of Nanomedicine, HST Center for Biomedical Engineering, Department of Medicine, Brigham and Women’s Hospital, ‡ Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, and § National Chemical Laboratories, Pune, India.  These authors contributed equally to this study. N anotechnology has been recog- nized by the National Cancer Insti- tute as a paradigm-changing op- portunity with the potential to enable significant breakthroughs in cancer diagno- sis and therapy. 1 The promise of cancer nano- technology lies in the ability to engineer customizable nanoscale constructs that can be loaded with one or more payloads such as chemotherapeutics, imaging, and diag- nostic agents. 2 However, nanocarriers widely explored to date such as liposomes, 3 polymers, 4 and dendrimers 5 suffer from in- herent limitations such as instability, struc- tural heterogeneity, synthetic challenges, and poor control over shape and size. Hence, there is an increasing need for ad- vanced delivery agents where shape, size distribution, functionalization, and loading capacity can be precisely tuned, with the as- sumption that a greater control over the physical parameters of the nanodevices may allow tunability of their biological fate. 6 The potential of fullerene to address the above design specifications lies in the im- mense scope for chemical derivatization to the basic structure. 7 In recent years, carbon nanotechnology has evolved into a truly in- terdisciplinary field, bridging material sci- ence with medicine. However, among the various currently available carbon nano- materials, it is the carbon nanotube (CNT), which has been mainly explored for bio- medical applications such as in the delivery of drugs or as probes for imaging. 8,9 In con- trast, fullerenes, the third allotrope of car- bon after diamond and graphite, have had limited use in biology due to their inherent hydrophobicity. The recent discovery that water-soluble fullerene derivatives can cross cell membranes 10 has accelerated in- terest in biological applications of C 60 11-16 such as in gene delivery. 12 Endohedral met- allofullerenes have demonstrated potential as radiopharmaceuticals 17 and MRI contrast agents. 18 However, the application of fullerenes as carriers of chemotherapeutics is still in a very nascent stage. There are only a few reports in the literature where fullerene derivatives have been used for the delivery of anticancer therapeutics. 19,20 Recently, an amphiphilic fullerene (bucky- some) was explored as a vector for pacli- taxel and was found to be equiefficaceous to Abraxane, a commercially available albumin-bound nanoformulation of pacli- taxel. 21 In the present study, we explored the novel application of polyhydroxylated fullerene (fullerenol) in drug delivery. The high aqueous solubility, neutral pH of fullerenols, and accessibility to further modification makes them promising candi- dates for medical applications. 22 Addition- ally, the facile synthetic procedure to pro- duce multiple hydroxyl groups 22 offers scope for high drug loading. We demon- strate that the fullerenols enable the *Address correspondence to shiladit@MIT.edu. Received for review March 30, 2009 and accepted August 07, 2009. Published online August 14, 2009. 10.1021/nn900318y CCC: $40.75 © 2009 American Chemical Society ABSTRACT In the present study, we report the novel application of polyhydroxylated fullerenes (fullerenols) in cancer drug delivery. The facile synthetic procedure for generating multiple hydroxyl groups on the fullerene cage offers scope for high drug loading in addition to conferring hydrophilicity. Doxorubicin, a first line cancer chemotherapeutic, was conjugated to fullerenols through a carbamate linker, achieving ultrahigh loading efficiency. The drugfullerenol conjugate was found to be relatively stable in phosphate buffer saline but temporally released the active drug when incubated with tumor cell lysate. The fullerenoldoxorubicin conjugate suppressed the proliferation of cancer cell-lines in vitro through a G2-M cell cycle block, resulting in apoptosis. Furthermore, in an in vivo murine tumor model, fullerenoldoxorubicin exhibited comparable antitumor efficacy as free drug without the systemic toxicity of free doxorubicin. Additionally, we demonstrate that the fullerenol platform can be extended to other chemotherapeutic agents, such as the slightly water-soluble cisplatin, and can emerge as a new paradigm in the management of cancer. KEYWORDS: fullerenol · nanoparticle · chemotherapy · doxorubicin · drug delivery ARTICLE www.acsnano.org VOL. 3 ▪ NO. 9 ▪ 2505–2514 ▪ 2009 2505