Investigation of Hexadecylphosphocholine (miltefosine) usage in Pegylated liposomal doxorubicin as a synergistic ingredient: In vitro and in vivo evaluation in mice bearing C26 colon carcinoma and B16F0 melanoma Manouchehr Teymouri a , Hamidreza Farzaneh b , Ali Badiee b , Shiva Golmohammadzadeh b , Kayvan Sadri c , Mahmoud Reza Jaafari a, ⁎ a Biotechnology Research Center, Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad 91775-1365, Iran b Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad 91775-1365, Iran c Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad 91775-1365, Iran abstract article info Article history: Received 20 June 2015 Received in revised form 10 August 2015 Accepted 17 August 2015 Available online 20 August 2015 Chemical compounds studied in this article: Doxorubicin Hydrochloride (PubChem CID: 443939) Miltefosine (PubChem CID: 3599) Cholesterol (PubChem CID: 5997) 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (PubChem CID: 64965) Keywords: Hexadecylphosphocholine Miltefosine Pegylated liposomes Chemotherapy Doxorubicin In this investigation, Hexadecylphosphocholine (HePC, miltefosine) was being used as a new ingredient in Pegylated liposomal doxorubicin (PLD) and different aspects of this integration such as its effect on doxorubicin (Dox) release and cell uptake, cytotoxicity of liposomes, in vivo distribution and half-life clearance time of Dox as well as median survival time were illustrated. The liposomal formulations were Pegylated liposomal doxorubicin containing 0, 0.5, 1, 2 and 4% mole ratios of HePC (HePC–PLD) and their respective Dox-free liposomes (HePC– PLs). The cells used were colon carcinoma (C26), adriamycin-resistant breast cancer (MCF-7-ADR), and B16F0 melanoma cell lines, of which C26 and B16F0 cells were exploited for tumoring in BALB/c and C57Bl/6 mice, respectively. In most cases, increase in miltefosine percentage resulted in physically liposomal instability, increased Dox delivery and toxicity and reduced blood half-life of Dox. Overall, HePC 4% –PLD and PLD differed significantly in many respects and it was considered too toxic to be injected at the same dose (15 mg Dox/ kg) as PLD. Although HePC 2% –PLD could extend the median survival time marginally in comparison to PLD, the concept of HePC- containing liposomes merits further investigation. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Elimination of cancerous cells with almost no or less harmful side ef- fects on normal tissues is the main concern with chemotherapy of can- cerous diseases. In fact, almost all chemotherapy schedules regardless of either the regimen that are used or the kind of drug that are virtually considered toxic, ultimately failed because of discretionary limitation of drug dose (Boranic and Raci, 1979; Nakazawa et al., 2001; Schumacher, 1999; Webber et al., 2007). As a result, chance of evolving resistant cancerous cell population increases, which in turn, results in malignancy (Gottesman, 2002; Piccart-Gebhart et al., 2005). Regarding this notorious fact, a majority of researchers are trying to find out methodologies for restricting serious side effects of chemotherapeutic agents and targeting cancerous cells (Dark et al., 1997; Huber et al., 1994) and their exclusive environment (Peer et al., 2007). One promising approach has already been proved that could be achieved by exploiting delivery systems ranging from 1–100 nm in size, in which large amount of drug could be accommodated (Davis et al., 2008; Peer et al., 2007). Among such delivery systems, Pegylated liposo- mal doxorubicin (PLD, commercially known as Doxil) is the cheap but in- telligent system with comparatively enhanced therapeutic effect (Wigler et al., 2002). There are several positive points in PLD that explain why PLD administration results in relatively less adverse side effects and more therapeutic value as opposed to Dox. Firstly, particles of such size exclu- sively accumulated in solid tumors. Secondly, the long chains of polyeth- ylene glycol polymers create a hydration shell around liposomes that protect them from endocytosis by reticuloendothelial system; therefore, avoiding hepatic engagement and hepatotoxicity, and extending plasma half-lives of liposomes. As a result, the opportunity for their passive accu- mulation in solid tumors is provided, where the microvessels are leakier European Journal of Pharmaceutical Sciences 80 (2015) 66–73 ⁎ Corresponding author. Tel.: +985138823255; fax: +985138823251. E-mail address: jafarimr@mums.ac.ir (M.R. Jaafari). http://dx.doi.org/10.1016/j.ejps.2015.08.011 0928-0987/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps