br Introduction br Recently paclitaxel PTX preparations whic
Recently, paclitaxel (PTX) preparations, which are eﬀective for the treatment of various tumors such as breast cancer, non-small cell lung cancer, and ovarian cancer have been widely used and studied [1,2]. This anticancer agent exerts a tumor eﬀect by polymerizing tubulin monomers into microtubules and stabilizing microtubules . Various formulations are used to improve the solubility of PTX, and Cremophor EL® (CrEL) used in Taxol® is a typical solubilizing agent. However, serious clinical side eﬀects, including hypersensitivity, nephrotoxicity, and neurotoxicity which are caused by PTX itself and CrEL are major obstacles for chemotherapy with PTX (1, 4–6).
Encapsulating PTX in a biocompatible carrier without using CrEL is useful for avoiding side eﬀects. Although biodegradable polymers and liposomes are frequently used as carriers of therapeutic agents for cancer treatment [4–10], we have focused on micelle preparations using amphiphilic compounds with high biocompatibility as a carrier for PTX. It is known that the pH around tumor cells is lower than that of normal cells [11,12]. Micelle preparations using pH-responsive
polymers have been extensively studied , and flower-like micelles , application to siRNA drugs , and utilization of amphiphilic peptide  have been reported. In our previous study, we synthesized amphiphilic compounds for micelle formulations. Highly reactive phosphoryl chloride, methyl lactate, and polyethylene glycol were used for synthesis, and alkyl side chains were introduced . The use of phosphoesters has drawn attention in biological and pharmaceutical applications because of the biocompatibility of poly(phosphoester)s and their structural similarity to teichoic PR-619 [18,19]. However, few re-ports are available on the application of phosphoesters to micelle for-mulations . pH-responsive carriers using poly(lactic acid) or poly (phosphoester) have been reported [14,21]. Micelle formulations using these carriers are expected to eﬃciently increase drug eﬃcacy by promoting drug release when reaching the periphery of the tumor. A formulation for cancer treatment is assumed to be administered re-peatedly. Hence, to avoid as much as possible the accelerated blood clearance phenomenon, which alters pharmacokinetic behaviors in se-rial injections [22–25], we used methoxypolyethylene glycol 350 (CH3O(CH2CH2O)nH, with a nominal molecular weight of 350 (Sigma-
Corresponding author at: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641, Yamazaki, Noda, Chiba 278-8510, Japan. E-mail address: [email protected] (K. Makino).
I. Takeuchi and K. Makino
Aldrich, St. Louis, MO) . Phosphorus-based polymers with alkyl side chains have been studied and they are being actively investigated for potential pharmaceutical and biomedical applications, such as car-riers of drug [26–29] and genes [30–33]. Since the hydrophobicity of the compound and the structure of the micelle change depending on the type of side chain, we synthesized four types of alkyl di(MePEG-lactate) phosphates with 1-dodecanol, 1-hexadecanol, 1-octadecanol, or 1-ei-cosanol as a side chain to select a compound suitable for encapsulation of PTX .
In this study, we prepared PTX-encapsulated micelles using four kinds of alkyl di(MePEG-lactate) phosphates. The structural formula of alkyl di(MePEG-lactate) phosphate is shown in Fig. 1 . We prepared micelles using these compounds and determined optimal compounds for cancer treatment based on their physicochemical properties. The degradability of the compound was evaluated at diﬀerent pH levels. In vitro hemolysis and cytotoxicity tests were performed on the selected micelles. In addition, their eﬀectiveness in cancer treatment was as-sessed by biodistribution study in tumor-bearing mice.
2. Materials and methods
PTX (C47H51NO14, purity > 98.0%) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Dimethyl sulfoxide (purity ≥99.0%), pyrene (purity ≥97.0%), polysorbate 80, and tetra-hydrofuran (THF; C4H8O, purity ≥99.5%, water ≤0.001%) were pur-chased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Phosphotungstic acid hydrate was purchased from Alfa Aesar (Ward Hill, MA). Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA). Ampicillin sodium was purchased from Meiji Seika Pharma Co., Ltd. (Tokyo, Japan). Dulbecco’s modified Eagle medium (DMEM; containing 1.0 g/L glucose, with L-glutamine and so-dium pyruvate) and CrEL were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Isoflurane for the animal was purchased from Mylan Inc. (Pittsburgh, PA). All other chemicals were of the highest grade commercially available.