Development of a liposomal formulation of the natural flavonoid fisetin
Graphical abstract
Development and optimization of a fisetin liposomal formulation that retains cytotoxic and morphological effects of free fisetin on endothelial cells.
Introduction
Several edible plant-derived compounds have been linked to the chemoprevention and treatment of cancer (Surh, 2003). Among these natural compounds, the flavonoids have shown several pharmacological properties of interest in the prevention and treatment of cancer (Havsteen, 2002, Lopez-Lazaro, 2002, Middleton et al., 2000).
In a program aimed at finding novel antiangiogenic agents, we have recently identified the natural flavonoid fisetin (3,3′,4′,7-tetrahydroxyflavone) as an interesting lead that can stabilize endothelial cells in vitro at non cytotoxic concentrations (Touil et al., 2009). Fisetin is present in several fruits, vegetables, nuts and wine (Arai et al., 2000, Kimira et al., 1998), and displays a variety of biological effects including antioxidant, anti-inflammatory (Park et al., 2007, Woodman and Chan, 2004), anti-carcinogenic and in vitro antiangiogenesis (Fotsis et al., 1997). Fisetin has been shown to inhibit several molecular targets, including cyclin-dependent kinases (Lu et al., 2005a, Lu et al., 2005b, Sung et al., 2007), DNA topoisomerases I and II (Constantinou et al., 1995, Olaharski et al., 2005), urokinase (Jankun et al., 2006), actin polymerization (Böhl et al., 2007), and androgen receptor signalling (Khan et al., 2008a).
Although fisetin has recently been shown to possess interesting anticancer activity in vivo in mice bearing lung carcinoma (Touil et al., 2010) and prostate tumours (Khan et al., 2008a), its in vivo administration remains problematic due to the relative water insolubility of this compound (Guzzo et al., 2006). To facilitate its in vivo administration, we therefore chose to formulate fisetin into liposomes and to evaluate if the new formulation retains the biological properties of fisetin.
Liposomes are artificial vesicles, composed of lipidic amphiphiles, usually phospholipids, which organise themselves in water to form an aqueous core surrounded by a lipidic bilayer. This structure allows liposomes to transport both hydrophilic and lipophilic compounds and have led to their clinical use as drug carriers of several drug classes including antibiotics, antifungals and anticancer agents (Allen and Cullis, 2004, Langer, 1998).
Concerning the anticancer agents, liposomes have been shown to allow a higher tumour accumulation of the drug (Gabizon, 1992). This tumour retention effect is apparently due to the liposomal drug extravasation through the tumour porous capillary endothelium, which appears to be a consequence of the rapid angiogenesis occurring in tumours and not in normal tissues (Yuan et al., 1995). Several liposomal forms of anthracyclines are currently employed in the clinic and these formulations have contributed to significantly reduce toxicity while maintaining their anticancer activity in breast (Batist et al., 2001, O’Brien et al., 2004) and soft tissue carcinoma (Siehl et al., 2005).
Hydrophobic compounds like flavonoids are not frequently formulated as liposomes, because a rapid exchange of the compounds may occur between the liposomal membrane and the cellular membrane (Fahr et al., 2006). However, this pharmaceutical formulation has recently been shown to improve the solubility of the flavonoid quercetin while maintaining its cytotoxicity in vitro, increase its blood residence time and its in vivo anticancer activity in mice (Yuan et al., 2006).
In the present study, our aim was therefore to develop and optimize a liposomal formulation of the flavonoid fisetin to facilitate its in vivo administration. We report here the different steps investigated to efficiently and easily encapsulate fisetin, while reducing the costs of the preparation by the assessment of the lipid/fisetin ratio, the choice of the liposomal method of preparation, and the excipient composition. Stability and in vitro cytotoxicity studies are also presented.
Section snippets
Chemicals
Fisetin, cholesterol, DMSO, Hepes and phosphate buffers have been purchased at Sigma–Aldrich. Phospholipids such as dioleoyl-phosphatidylcholine, distearylphosphatidylethanolamine-poly(ethyleneglycol)2000 have been purchased from Aventi-Polar, Inc., and P90G has been obtained from Lipoid. Dichloromethane, triethylamine, chloroform, methanol, absolute ethanol and silica gel were provided by Carlo Erba Reactif, SDS. BOP was purchased at Advanced ChemTech. CHOL-PEG2000(OH) was synthesized as
Determination of the maximum amount of fisetin inserted in the lipid bilayer
Fisetin is a lipophilic compound that solubilises into the lipidic bilayer because its log P octanol–water is estimated to be about 3.2 (Miteva et al., 2006). We initially evaluated the interaction of fisetin with dimyristoylphosphatidylcholine as solubilizer (Guharay et al., 2001), then evolved to more sophisticated liposome formulation to increase fisetin stability into the bilayer. Obviously, inserting a too large amount of fisetin would induce a destabilisation of the bilayer, therefore
Acknowledgements
This research was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), by the Centre National de la Recherche Scientifique (CNRS) and by a grant from the Institut National du Cancer (INCa, Boulogne-Billancourt, France). We also thank René Lai-Kuen and Bruno Saubamea from Technical Platform of the IFR71/IMTCE–Cellular & Molecular Imaging–Faculty of Pharmacy–Paris Descartes University, and Eric Larquet from the IMPMC, CNRS-UMR7590, Université Pierre & Marie Curie,
References (44)
- et al.
Dietary intakes of flavonols, flavones and isoflavones by Japanese women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration
J. Nutr.
(2000) - et al.
Ferrier rearrangement for the synthesis of PEG-bound 2,3-unsaturated glycopyranosyl-amino acids
Tetrahedr. Lett.
(2007) - et al.
Flavonoids affect actin functions in cytoplasm and nucleus
Biophys. J.
(2007) The biochemistry and medical significance of the flavonoids
Pharmacol. Ther.
(2002)- et al.
Relationship between molecular structure and supramolecular morphology of DODA-EO2-biotin and related lipids
Chem. Phys. Lipids
(1997) - et al.
Fisetin inhibits the activities of cyclin-dependent kinases leading to cell cycle arrest in HT-29 human colon cancer cells
J. Nutr.
(2005) - et al.
pH-sensitive PEG lipids containing orthoester linkers: new potential tools for nonviral gene delivery
J. Controlled Release
(2004) - et al.
Anionic polyethyleneglycol lipids added to cationic lipoplexes increase their plasmatic circulation time
J. Controlled Release
(2003) - et al.
Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer
Ann. Oncol
(2004) - et al.
Chromosomal malsegregation and micronucleus induction in vitro by the DNA topoisomerase II inhibitor fisetin
Mutat. Res./Genet. Toxicol. Environ. Mutagen.
(2005)