Synergistic anti-tumor activity of paclitaxel-incorporated conjugated linoleic acid-coupled poloxamer thermosensitive hydrogel in vitro and in vivo
Introduction
Paclitaxel (PTX) is one of the most successful anticancer drugs found in the past decades. It has been clinically used in the treatment of a wide variety of cancers like breast, ovarian, skin, and lung cancers [1]. However, PTX has a low therapeutic index due to highly lipophilic and virtually insoluble properties in water [2]. An adjuvant consisting of Cremophor EL (polyethoxylated castor oil) and dehydrated alcohol is used in the current clinical administration of PTX and causes serious side effects including neurotoxicity, hypersensitivity reactions, and nephrotoxicity [3], [4]. To overcome the problems caused by Cremophor EL, and to improve the drug efficacy, recent research has been focused on developing new drug delivery systems. Several new formulations of PTX are in clinical trials or marketed. Abraxane®, an albumin-bound PTX, is approved for use in patients with metastatic breast cancer who fail combination chemotherapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy [5]. Xyotax®, poly(l-glutamic acid)–PTX, enhances tumor exposure by taking advantage of the hyperpermeability of tumor vasculature and suppressed lymphatic clearance characteristic of tumor tissue [6]. And poly(lactic acid) (PLA) based nanoparticles are under preclinical research [7], [8]. A variety of approaches have been investigated including liposomes [9], polymer micelles [10], polymeric biodegradable nanoparticles [2], [11], [12], water-soluble pro-drugs [13], [14], albumin conjugates [15], and hydrogels [16], [17], [18]. A novel alternate is the development of an injectable formula, hydrogel, which provides high local concentration of the drug and prevents the systemic side effects of the chemotherapy normally associated with the intravenous administration.
Here, we report on the development of a thermosensitive conjugated linoleic acid (CLA)-coupled poloxamer hydrogel as a carrier for local delivery of PTX. Poloxamer consists of hydrophilic ethylene oxide (EO) and hydrophobic propylene oxide (PO) blocks, arranged in a basic structure of EOa–POb–EOa [19]. It has been widely exploited as an in-situ forming drug delivery carrier because it exhibits unique sol–gel transition behaviors in response to temperature in aqueous solution [20]. These copolymers form spherical micelles in aqueous solution by hydrophobic interaction between the middle PPO segments [21]. Above a critical gelation temperature and concentration, the self-assembled micelles pack closely to produce a physically crosslinked gel structure.
Conjugated linoleic acid (CLA) refers to a group of polyunsaturated fatty acids (PUFAs) that exist as positional and stereoisomers of octadecadienoic acid. Several studies have shown that the PUFAs seemed to sensitize tumor cells to chemotherapy and radiotherapy in cell culture, in tumor-bearing animals, and in humans [22], [23], [24], [25]. We previously reported that CLA-coupled poloxamer had enhanced anti-tumor activity compared to CLA [26]. Also, the CLA-coupled poloxamer formed a more stable hydrogel than poloxamer itself due to the hydrophobic property of CLA, even at a low concentration. We hypothesized that the CLA-coupled poloxamer would increase the efficacy of the cancer chemotherapeutic agent, PTX, because solubility of the PTX would be increased by the CLA-coupled poloxamer micelles, and the anticancer agent CLA would be released from the CLA-coupled poloxamer as the pro-drug. The anti-tumor activity of PTX-incorporated CLA-coupled poloxamer was assessed with breast cancer cell lines in vitro and with breast cancer xenografts in vivo.
Section snippets
Materials
CLA was kindly provided by HK Biotech (Seoul, Korea) and contained 45.98% cis-9, trans-11 and 49.88% trans-10, cis-12 CLA. Poloxamer (Pluronic F127) (Mw: 12,600) was provided by BASF Korea Inc. (Seoul, Korea) and was used without further purification. PTX was purchased from Samyang Genex Corporation (Daejeon, Korea).
Cell lines and cell culture
MDA-MB-231 and MCF-7 human breast cancer cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA). MDA-MB-231 and MCF-7 human breast cancer cells
Characteristics of CLA-coupled poloxamer and PTX solubility in CLA-coupled-poloxamer formulation
CLA-coupled poloxamer was successfully synthesized through ester linkage between the carboxyl group of CLA and the hydroxyl group of poloxamer as shown in Fig. 1A. The composition of CLA in the synthesized CLA-coupled poloxamer estimated by 1H NMR spectroscopy was 49.2 mol%. Due to the thermogelling property of poloxamer, CLA-coupled poloxamer also showed this thermogelling property at even low concentration compared with poloxamer itself due to the hydrophobic property of CLA (data not shown).
Discussion
Regional delivery of anti-tumor drugs is expected to provide a high local concentration thereby decreasing the incidence of side effects commonly observed with systemic therapy [30]. Hydrogel systems can be used as a delivery system directly by administration into a mouse body or tumor tissue [31], [32]. In this study, we prepared a thermosensitive CLA-coupled poloxamer hydrogel containing a chemotherapeutic agent, PTX, for local delivery of PTX. We predicted that the CLA-coupled poloxamer
Conclusion
In this study, a CLA-coupled poloxamer thermosensitive hydrogel was used as a local delivery system for PTX. The CLA-coupled poloxamer significantly increased the PTX anticancer efficacy through induction of the cell cycle arrest and apoptosis in breast cancer treatment both in vitro and in vivo. Therefore, use of thermosensitive CLA-coupled poloxamer hydrogel as a carrier in the formulation of chemotherapeutic drugs may be beneficial for the treatment of various cancers.
Acknowledgements
This work was supported by the Agricultural R&D Promotion Center (ARPC) (2006-0053). National Instrumentation Centre for Environmental Management (NICEM) is acknowledged for providing analytical facilities.
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- 1
These authors contributed equally to this work.
- 2
Present address: Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA.