Research paper Methotrexate loaded chitosan nanoparticles: Preparation, radiolabeling and in vitro evaluation for breast cancer diagnosis Meliha Ekinci * , Derya Ilem-Ozdemir, Evren Gundogdu, Makbule Asikoglu Faculty of Pharmacy, Department of Radiopharmacy, Ege University, 35100 Bornova, Izmir, Turkey article info Article history: Received 9 September 2015 Received in revised form 30 September 2015 Accepted 2 October 2015 Available online 9 October 2015 Keywords: Methotrexate Chitosan nanoparticles Technetium-99m Radiolabeling Radiopharmaceuticals Breast cancer diagnosis abstract This study aimed to developed radiolabeled methotrexate (MTX) loaded nanoparticulate systems for breast cancer imaging. For this aim, two formulations (F1 and F2) of MTX loaded chitosan nanoparticles (CSNPs) were prepared via ionic gelation process by using CS and tripolyphosphate (TPP). The obtained results showed that by using F1 and F2 formulations with mean diameter of 169.000 nm and 427.633 nm, zeta potential of 20.133 mV and 29.067 mV were successfully developed with ionotropic gelation process and the encapsulation efficiency (EE) of F1 and F2 was found nearly 35% and 64% respectively. In addition, MTX-CSNPs were radiolabeled by Technetium-99m ( 99m Tc) and radiochemical purity and stability of labeled compound were performed using a gamma counter up to 6 h. Results indicated that MTX-CSNPs were radiolabeled by 99m Tc with high labeling efficiency (>90%) and stability. For in vitro incorporation studies the uptake differences between 99m Tc labeled MTX loaded and unloaded CSNPs, reduced/hydrolyzed (R/H) 99m Tc were evaluated in human breast cancer (MCF-7) and human keratinocyte (HaCaT) cell lines. According to cell culture studies, the incorporation percentages of 99m Tc-MTX-CSNPs were highly uptake in cancer cell line. The results demonstrated that radiolabeled MTX-CSNPs may be a promising agent for breast cancer diagnosis. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Breast cancer is the most common invasive cancer in women worldwide [1]. In cancer medicine, nanotechnology holds great promise to revolutionize drug delivery, gene therapy, diagnostics and many areas of research, development and clinical application [2]. In general, nanocarriers may protect drug from degradation, enhance drug absorption by facilitating diffusion through epithe- lium, modify pharmacokinetic and tissue distribution profile, and/ or improve intracellular penetration. Also nanosystems were found useful to improve the performance of imaging techniques applied for the in vivo diagnosis of tumors [3]. Nanoparticles (NPs) are materials with typically overall di- mensions less than several hundred nanometers and about 2e4 orders of magnitude smaller than human cells. Because of this unique physical property, NPs demonstrate marvelous interactions with both on surface and inside the cancer cells. Therefore, NPs have wide applications as targeted delivery agents in cancer diag- nosis and treatment [4,5]. Also unwanted effects of drugs on bystander cells and tissues can be minimizing by using NPs with appropriate particle size [6]. Polymeric NPs have shown preferential accumulation at tumor sites, their usage as carriers improves efficacy and reduces side effects. Chitosan (CS) has been widely used to prepare nano- particulate drug delivery system since has many good bioproperties and physiochemical characteristics. CS is a natural polysaccharide which derived from chitin by deacetylation. This cationic polymer is regarded as biocompatible, biodegradable and non-toxic. The cationic properties of CS are particularly valuable for drug delivery systems. For example, ion complexes between CS and anionic drugs (i.e. methotrexate (MTX)) can be formed to NPs [7e9]. Recent studies showed that in tumor tissue folate receptors were found higher compared with healthy tissue. The folate re- ceptors are overexpressed on a variety of human tumors, such as breast tumor, thus making folate receptors a potential molecular target for tumor imaging. MTX, is an analogue of folic acid, which exhibits not only a targeting role as folic acid but also a therapeutic effect to many types of cancer cells that overexpress folate re- ceptors on their surfaces. So it has been utilized for the treatment of * Corresponding author. E-mail addresses: meliha.ekinci@ege.edu.tr (M. Ekinci), deryailem@gmail.com (D. Ilem-Ozdemir), evren.gundogdu@ege.edu.tr (E. Gundogdu), makbule. asikoglu@ege.edu.tr (M. Asikoglu). Contents lists available at ScienceDirect Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst http://dx.doi.org/10.1016/j.jddst.2015.10.004 1773-2247/© 2015 Elsevier B.V. All rights reserved. Journal of Drug Delivery Science and Technology 30 (2015) 107e113