Colloids and Surfaces B: Biointerfaces 153 (2017) 320–326 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces jo ur nal ho me p ag e: www.elsevier.com/locate/colsurfb Protocols In vitro evaluation of cytotoxicity, possible alteration of apoptotic regulatory proteins, and antibacterial activity of synthesized copper oxide nanoparticles Shahanavaj Khan a,∗ , Anees A. Ansari b , Azmat Ali Khan c , Maha Abdulla d , Omar Al-Obaid d , Rehan Ahmad d a Nanomedicine & Biotechnology Research Unit, Department of Pharmaceutics, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia b King Abdullah Institute for Nanotechnology, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia c Pharmaceutical Biotechnology Laboratory, Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO Box 2455, Riyadh, Saudi Arabia d Colorectal Research Centre, College of Medicine, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia a r t i c l e i n f o Article history: Received 20 December 2016 Received in revised form 2 March 2017 Accepted 2 March 2017 Keywords: Copper oxide nanoparticles Colon cancer cell lines MTT assay Cytotoxicity Western blot a b s t r a c t Copper oxide nanoparticles (CuO-NPs) were synthesized using a urea-based thermal decomposition tech- nique, and characterized using different techniques. X-ray diffraction (XRD) and Raman spectroscopy confirmed the phase purity and crystalline structure of CuO-NPs. The size of CuO-NPs was investigated using XRD and was confirmed via dynamic light scattering analysis. CuO-NPs showed an average diameter of ∼20 nm. The possible cytotoxicity of CuO-NPs was evaluated in HT-29 and SW620 cancer cell lines. The median inhibitory concentration of CuO-NPs in HT-29 and SW-620 cells was 4.99 and 3.75 g/mL, respec- tively. The underlying mechanism responsible for apoptosis in colon cancer cells after CuO-NP exposure has not been well understood. In this study, we investigated the possible mechanisms of induction of apoptosis via analysis of the expression of Bcl-2 and Bcl-xL proteins in HT-29 human colon cancer cells after CuO-NP exposure. Western blot assay showed downregulation of Bcl-2 and Bcl-xL protein expres- sion after CuO-NP exposure. Our findings may aid in the understanding of the potential mechanisms responsible for induction of apoptosis owing to inhibition of Bcl-2 and Bcl-xL protein expression. Fur- thermore, the antibacterial activity assay showed that the synthesized CuO-NPs did not exert significant inhibitory effects against different gram-positive and gram-negative bacteria in vitro. © 2017 Elsevier B.V. All rights reserved. 1. Introduction Nanoparticles are the building blocks of nanomedicine and nanotechnology. In the field of medicine, nanobiotechnology has been used in various applications, such as detection, diagnosis, drug delivery, and treatment of various diseases, including different types of cancer [1–5]. The recent intensification toward the impli- Abbreviations: CuO-NPs, copper oxide nanoparticles; DLS, dynamic light scatter- ing; PDI, polydispersity index; XRD, X-ray diffraction; HT-29, human colon cancer cell line; SW-620, human colon cancer cell line; Bcl-2, B-cell lymphoma 2; Bcl-xL, B- cell lymphoma-extra large; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; MTT reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; E. coli, Escherichia coli; IC50, half maximal inhibitory concentration; ◦ C, degree celsius; g, microgram; mL, milliliter; h, hour. ∗ Corresponding author. E-mail address: khan.shahanavaj@gmail.com (S. Khan). cation of bioreactive nanoparticles helps to open a new era in the connection of nanotechnology with biotechnology. Various stud- ies have shown the potential applications of the novel molecular benign technologies for therapeutic and diagnostic purposes in the biological system by implication of nanotechnology [4,6]. Cancer is a leading cause of mortality worldwide. Despite the advances made toward the understanding of the molecular basis of cancer and the development of surgical procedures, chemother- apy, and radiotherapy, as well as the identification of several cancer biomarkers, the overall cancer death rate has not significantly improved since the last two decades [7]. The progress achieved in nanotechnology and nanobiotechnology has been widely consid- ered as a novel and revolutionary paradigm shift for diagnosis and treatment of different types of cancer. The design and synthesis of different types of nanoparticle (NP) systems using various materi- als may provide a promising approach for the treatment of various cancer types [3,5,8]. Therefore, more efforts are required to aid in http://dx.doi.org/10.1016/j.colsurfb.2017.03.005 0927-7765/© 2017 Elsevier B.V. All rights reserved.