Intranuclear Drug Delivery and Effective in Vivo Cancer Therapy via Estradiol-PEG-Appended Multiwalled Carbon Nanotubes Manasmita Das, Raman Preet Singh, Satyajit R. Datir, and Sanyog Jain* Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar (Mohali), Punjab 160062, India * S Supporting Information ABSTRACT: Cancer cell-selective, nuclear targeting is expected to enhance the therapeutic efficacy of a myriad of antineoplastic drugs, particularly those whose pharmacody- namic site of action is the nucleus. In this study, a steroid- macromolecular bioconjugate based on PEG-linked 17β- Estradiol (E 2 ) was appended to intrinsically cell-penetrable multiwalled carbon nanotubes (MWCNTs) for intranuclear drug delivery and effective breast cancer treatment, both in vitro and in vivo. Taking Doxorubicin (DOX) as a model anticancer agent, we tried to elucidate how E 2 appendage influences the cell internalization, intracellular trafficking, and antitumor efficacy of the supramolecularly complexed drug. We observed that the combination of DOX with E 2 -PEG- MWCNTs not only facilitated nuclear targeting through an estrogen receptor (ER)-mediated pathway but also deciphered to a synergistic anticancer response in vivo. The antitumor efficacy of DOX@E 2 -PEG-MWCNTs in chemically breast cancer-induced female rats was approximately 18, 17, 5, and 2 times higher compared to the groups exposed to saline, drug-deprived E 2 -PEG- MWCNTs, free DOX, and DOX@m-PEG-MWCNTs, respectively. While free DOX treatment induced severe cardiotoxicity in animals, animals treated with DOX@m-PEG-MWCNTs and DOX@E 2 -PEG-MWCNTs were devoid of any perceivable cardiotoxicity, hepatotoxicity, and nephrotoxicity. To the best of our knowledge, this is the first instance in which cancer cell- selective, intranuclear drug delivery, and, subsequently, effective in vivo breast cancer therapy has been achieved using estrogen- appended MWCNTs as the molecular transporter. KEYWORDS: cancer, intranuclear drug delivery, estrogen, carbon nanotubes, antitumor efficacy 1. INTRODUCTION Estrogen hormones, in particular, 17β-estradiol (E 2 ), have been identified as one of the most vital hormones regulating the development and maintenance of the female reproductive system and secondary sex characteristics. 1-3 Binding of E 2 with estrogen receptors (ER) induces conformational changes and release of molecular chaperone (Hsp 90, Hsp 70, cyclophilin, and p23) from the receptors, 4 which allows them to conscript the cofactors necessary for transcription of various genes commonly upregulated in malignant cells (e.g., transforming growth factor alpha, c-myc, or cathepsin D). As evident from an extensive literature survey, hormone receptors like ERs and progesterone receptors are overexpressed in 70-80% of all breast cancers. The upregulation of ERs in cancerous cells relative to normal cells can be effectively harnessed for the development of a targeted therapy against various hormone sensitive cancers. 5,6 ER-α is known to localize in both nucleus as well as plasma membrane, mediating estrogen-dependent, genomic, and nongenomic signaling. 4,7-10 Subsequently, conjugation of estrogen hormones with any pharmaceutically active component (either free or carrier bound) may simultaneously facilitate cellular and intracellular, organelle- specific (nuclear) targeting in such receptor overexpressed cancer cells. A large number of first line chemotherapeutic medications, including doxorubicin (DOX) and cisplatin (CDDP), exert their pharmacodynamic effects in nuclei by intercalating with DNA base pairs, thereby inhibiting cell growth/proliferation. 11 Unfortunately, the transport of these anticancer drugs from plasma membrane to nucleus is not very well-characterized. In fact, transport of drugs to nuclei have been found to be rather difficult, and even if it could happen, it is considered to be nonspecific and passive. Additionally, drug-resistant cancer cells have many intracellular drug-resistance mechanisms that avert the access of anticancer agents to the nucleus. Consequently, only a small percentage of the administered dose can be delivered into the cytosol and finally reach the nucleus. 9 Over the past years, a number of ER-targeted bioconjugates have been prepared by coupling estrogens with a myriad of cytotoxic Received: April 21, 2013 Revised: July 7, 2013 Accepted: August 1, 2013 Article pubs.acs.org/molecularpharmaceutics © XXXX American Chemical Society A dx.doi.org/10.1021/mp4002409 | Mol. Pharmaceutics XXXX, XXX, XXX-XXX