Journal of Drug Delivery Science and Technology 80 (2023) 104146 Available online 30 December 2022 1773-2247/© 2022 Elsevier B.V. All rights reserved. Radiolabeled methotrexate loaded chitosan nanoparticles as imaging probe for breast cancer: Biodistribution in tumor-bearing mice Meliha Ekinci a , Cinel Koksal-Karayildirim b , Derya Ilem-Ozdemir a, * a Faculty of Pharmacy, Department of Radiopharmacy, Ege University, 35040, Bornova, Izmir, Turkey b Faculty of Science, Department of Biology, Ege University, 35040, Bornova, Izmir, Turkey A R T I C L E INFO Keywords: Biodistribution Breast cancer Chitosan nanoparticles Methotrexate Radiolabeling Radiopharmaceuticals Technetium-99 m ABSTRACT There is great interest in developing nanoparticle drug delivery systems for the delivery of therapeutics or im- aging agents for cancer. In this context, a new radiopharmaceutical was prepared by labeling the methotrexate (MTX) loaded chitosan nanoparticles (CSNPs) with [ 99m Tc]Tc radionuclide, and the breast cancer imaging po- tential of the radiopharmaceutical was determined by in-vivo biodistribution studies. Within the scope, different amounts of MTX-loaded CSNPs (MTX-CSNPs) (F1–F3) were prepared via an ionotropic gelation process. The physicochemical properties of MTX-CSNPs were evaluated. The mean diameter of MTX-CSNPs was found be- tween 148.8 ± 0.636 nm and 157.0 ± 3.960 nm, the PdI value of between 0.415 ± 0.042 and 0.451 ± 0.036 and the zeta potential of between 15.7 ± 1.17 mV and 18.7 ± 0.27 mV. In addition, the preparation effciency of MTX-CSNPs was found to be between 64.036 ± 2.054% - 70.234 ± 2.140% and the encapsulation effciency of CSNPs was found to be between 32.301 ± 1.205–50.304 ± 2.478%. MTX-CSNPs were radiolabeled with [ 99m Tc] Tc, and the radiolabeled nanoparticles ([ 99m Tc]Tc-MTX-CSNPs) were evaluated by both in-vitro cell binding studies and in-vivo biodistribution studies. [ 99m Tc]Tc-MTX-CSNPs showed high labeling effciency (≥99%) and in-vitro stability up to 6 h at room temperature. According to the cell incorporation study, [ 99m Tc]Tc-MTX-CSNPs (F3) showed signifcantly higher uptake into 4T1 (breast cancer) cells than R/H-[ 99m Tc]Tc. Biodistribution studies of [ 99m Tc]Tc-MTX-CSNPs (F3) were performed in breast tumor-bearing female Balb/c mice. Compared to [ 99m Tc]Tc-MIBI, [ 99m Tc]Tc-MTX-CSNPs (F3) exhibited signifcantly higher uptake by breast tumors. This study demonstrates the affnity of [ 99m Tc]Tc-MTX-CSNPs to breast cancer cells and their diagnostic utility. 1. Introduction Since breast cancer is the most common invasive cancer in women worldwide, early diagnosis is an important advantage for breast cancer patients [1]. Before starting breast cancer treatment, the diagnosis and staging of breast cancer should be done accurately and effectively [2]. With the development of diagnostic possibilities, more cancer cases are diagnosed every year [3,4]. Although mammography and ultrasonog- raphy are the most used imaging methods in breast cancer screening and diagnosis [5], benign and malignant lesions may appear similar in both methods. These anatomical imaging techniques are not suffcient for imaging in the frst stage of the disease because they are based on morphological changes. Scintigraphic imaging techniques [Positron Emission Tomography (PET), single photon emission computed to- mography (SPECT)], on the other hand, allow diagnosis at an early stage, as it is a non-invasive imaging technique based on the detection of physiological changes [6]. [ 99m Tc]Tc-labeled 2-methoxy isobutyl iso- nitrile (MIBI) and Fluor–18-labeled fuoro-2-deoxy-D-glucose ([ 18 F] FDG) are routinely used radiopharmaceuticals for clinical screening of breast cancer [7,8]. However, as it is known, these radiopharmaceuti- cals are not specifc for imaging breast cancer, and new imaging agents are needed for early diagnosis. In recent years, with the increase in molecular imaging studies, a large number of studies have begun to be conducted on the use of radiolabeled nanostructured radiopharmaceuticals for diagnostic, ther- apeutic or theranostic purposes [9–11,12]. These preclinical studies, especially in the feld of cancer, play an important role in the develop- ment of new tumor-specifc nanobioradiopharmaceuticals. In this context, in our study, a new nanostructured radiopharmaceutical was developed to be used in the diagnosis of breast cancer. * Corresponding author. E-mail addresses: melihaekinci90@gmail.com (M. Ekinci), cinel.koksal.karayildirim@gmail.com (C. Koksal-Karayildirim), deryailem@gmail.com (D. Ilem- Ozdemir). Contents lists available at ScienceDirect Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst https://doi.org/10.1016/j.jddst.2022.104146 Received 26 August 2022; Received in revised form 25 December 2022; Accepted 30 December 2022