Delivered by Publishing Technology to: University of Gazi IP: 194.27.18.18 On: Tue, 19 Aug 2014 07:02:19 Copyright: American Scientific Publishers Copyright © 2012 American Scientific Publishers All rights reserved Printed in the United States of America Journal of Biomedical Nanotechnology Vol. 8, 508–514, 2012 A Novel Protein-Based Anticancer Drug Encapsulating Nanosphere: Apoferritin-Doxorubicin Complex Mehmet A. Kilic 1 ∗ , Erdal Ozlu 1 , and Sema Calis 2 1 Akdeniz University, Science and Arts Faculty, Department of Biology, Molecular Biology Section, 07058, Antalya, Turkey 2 Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100, Ankara, Turkey Developing a drug delivery system, which is uniform, biocompatible, stable and non-toxic, is a challenging issue in anticancer drug delivery strategies. Ferritin is a nano-size spherical protein with an internal cavity where drug molecules can be encapsulated. The apoferritin-doxorubicin complex has been formed by ‘opening’ and ‘closing’ the apoferritin sphere in the presence of doxorubicin. The doxorubicin encapsulation was carried out using direct and step-wise change of pH of the solution from 2.5 to 7.4. Non-denaturing polyacrylamide gels showed that the protein cage of the complex successfully self-assembles into its nanosphere form. It was found that up to 28 molecules of doxorubicin can be capsulated per apoferritin protein and no significant drug leakage occurs during the first two days. The apoferritin-doxorubicin complex is a promising nanocarrier for the delivery of anticancer drugs. Keywords: Ferritin, Doxorubicin, Nanosphere, Drug Encapsulation, Drug Delivery, Cancer Therapy. 1. INTRODUCTION Developing an anticancer drug carrier that will effec- tively deliver chemotherapeutics to cancer cells is a major challenge in cancer therapy strategies. Several anticancer drug encapsulation and delivery systems, liposomes, 1 2 biodegradable microspheres, 3 antibodies, 4 dendritic polymers 5 and protein-based carriers 6 7 are being developed to deliver drugs to neoplastic tissue and organs. Ferritins are composed of 24 polypeptide subunits packed together to form a nanosphere of diameter ∼12 nm, with an internal cavity ∼8 nm across, where up to 4,500 Fe(III) atoms are stored. 8 Mammalian ferritins are composed of two subunits, namely light (L) and heavy (H) chains with respective molecular weights of 19 and 21 kDa. 9 Ferritin proteins, in addition to their well-known cytoplasmic iron storage function, deliver iron to organs and play a key role in the regulation of the iron status of tissues and organs. 10 In humans, ferritin exists in tissue as well as in serum and the serum ferritin is internalized by their specific receptors. 11 Several normal and cancer cell lines express ferritin receptors and their numbers are higher in cancer cells. 12 Ferritin can also pass the blood ∗ Author to whom correspondence should be addressed. brain barrier with its specific receptor and deliver iron to the brain. 13 These unique properties of ferritin proteins have recently attracted the attention of several researchers to investigate their potential as anticancer drug (platinum and variants) encapsulation systems, 14 15 16 cancer cell target- ing magnetic nanoparticles, 17 quantum dot carriers for can- cerous cells 18 and label and contrast agent carriers. 19 20 Doxorubicin is a commonly used chemotherapeutic agent for the treatment of a variety of cancers. However, the systemic toxicity of doxorubicin, and in particular its asso- ciation with the development of substantial cardiotoxicity, limits its effective usage in cancer therapy. 21 Encapsulating doxorubicin in liposomal vesicles has significantly reduced the cardiotoxicity of the drug in cancer patients 22 and targeting liposome-doxorubicin conjugates has improved its efficacy in tumor models. 23 Doxorubicin encapsulation within the cavity of the highly stable monodisperse fer- ritin cage could reduce the general toxicity of free drug molecules and improve the efficacy of the drug by deliv- ering its cargo to tumor tissues. The anticancer drug deliv- ery potential of horse spleen apoferritin has recently been explored and it has been found that the ferritin can deliver platinum-based drugs to the rat cancer cell line and result in cytotoxicity. 16 508 J. Biomed. Nanotechnol. 2012, Vol. 8, No. 3 1550-7033/2012/8/508/007 doi:10.1166/jbn.2012.1406