Cytotoxic effect of novel Flammulina velutipes sterols and its oral bioavailability via mixed micellar nanoformulation

Abstract

The aim of this study was to investigate the anti-tumor effect of sterols initially separated from Flammulina velutipes and the pharmacokinetics and tissue distribution after oral administration of F. velutipes sterol nanomicelles (FVSNs). F. velutipes sterol (FVS) consisted of mainly ergosterol (54.78%), 22,23-dihydroergosterol (27.94%) and ergost-8(14)-ene-3β-ol (discovered for the first time in F. velutipes). In vitro cytotoxicity assay of FVS against U251 cells and HeLa cells showed that at 72 h treatment, the FVS (IC₅₀ = 23.42 μg/mL) exhibited strong inhibitory effect against U251 cells, even overwhelmed the standard anti-tumor drug (5-fluorouracil) to an extent, while the HeLa cells were not significantly susceptible to the FVS. To improve the solubility and bioavailability of FVS, a model for insoluble anti-tumor drugs, FVSNs were prepared. In vitro characterization of FVSNs revealed satisfactory size distribution, loading capacity and encapsulation efficiency. Pharmacokinetic study in SD rats demonstrated that the mixed micellar nanoformulation significantly enhanced the bioavailability of FVS than free drug. Additionly, tissue distribution in mice manifested that the biodistribution of FVSNs as compared to the free FVS suspension were significantly improved. In conclusion, the nanomicelles developed in our study provided a promising delivery system for enhancing the oral bioavailability and selective biodistribution of FVS, a potential anti-tumor agent.

Introduction

Enormous efforts are ongoing to identify new anticancer compounds from plants, no wonder in eastern countries, China especially, Traditional Chinese medicine (TCM) occupied, and still occupies a significant position in primary health care in less developing rural areas and also greatly appreciated in well developed urban due to its thousands-year-old clinical tradition, or in the West, interest in TCM stems from the hope that it might complement Western medicine and bioactive natural products as chemical lead compounds for the generation of semi-synthetic derivatives. However, the unclear multi-components and therapeutic mechanism, limited water solubility and bioavailability as well as poor targeting specificity greatly hamper the drug ability of these promising antitumor candidates. Naturally, of particular interests are technologies for improving the bioavailability and targeted delivery with the administration of these compounds. Of further interest is the elucidation of the assessment of their kinetics, tissue distribution and elimination routes. These data have indeed become an important issue to link data from pharmacological assays and clinical values. Besides, a better understanding of the pharmacokinetics and bioavailability of phytopharmaceuticals can also aid in designing rational dosage regimens (Bhattaram et al., 2002).

Flammulina velutipes, belonging to Basidiomycotina of Eumycota, popularly known as golden-needle mushroom or winter mushroom, is a kind of nutritional and vegetable functional food with high edible value for its rich content in proteins and carbohydrates, high in fiber and low in fat (Ko et al., 2007, Yang et al., 2007). With the polysaccharides, proteins and glycoproteins as the important components isolated, F. velutipes have been correlated with multiple pharmacological activities such as immunomodulatory, antioxidant, cholesterol-lowering, hypoglycaemia-induction and anti-cancer activities (Ko et al., 1995, Leung et al., 1997, Wu et al., 2008, Yang et al., 2012). However, when it comes to small molecules, the information on biological activities is limited, which is well proven by the scarce studies on F. velutipes sterol (FVS). To our best knowledge, no research has been reported on the in vitro antineoplastic activity of FVS in the treatment of gliomas and cervical carcinoma up to now.

Given the chemical property, FVS can easily dissolve in ether, chloroform, hot ethanol as well as other organic solvents, but to a lesser extent in water, as a result its future utilization in the clinical area would be discounted extremely heavy. In recent years, advances in nanotechnology (polymeric nanoparticles or micelles, nanocapsules, liposomes, carbon nanotubes, phytosomes and microemulsion etc.) have shown promise in improving component solubility, enhancement of bioavailability, increasing absorbency of the organism, reducing medicinal herb doses, together with achieving steady-state therapeutic levels of drugs over an extended period compared with traditional Chinese herb drug preparations (Ajazuddin and Saraf, 2010, Bhadoriya et al., 2011, Yang et al., 2010). Among these nanocarriers, nanomicelles have emerged as one of the most useful modalities for suitable and versatile drug carriers. The polymeric micelles are self-assembling nanoscale systems and formed by microphase separation in selective solvents. Its unique features including hydrophilic outer shell and hydrophobic inner core impart its ability to dissolve in water, which leads to the spontaneous formation of nanomicelles with the competency of encapsulating and solubilizing compounds of different nature. Through a sterol-loaded microemulsion formulation, our previous study (Yi et al., 2012) succeeded in enhancing oral bioavailability of FVS. However, no significant improvement appeared in the data of elimination half-life (t_1/2) of sterol encapsulated in microemulsions compared with free sterol, suggesting its less-than-ideal duration of pharmacologic action. Comparatively, self-assembled nanomicelles, composed of polymeric amphiphiles, have a potential to bring several advantages to therapeutic systems because of the long circulation time, high drug loading capacity and low dose of formulation needed (Ajazuddin and Saraf, 2010, Gong et al., 2012, Jiang et al., 2011, Muthu et al., 2009). Besides, compared with long-circulating liposomes, nanomicelles are of some unique merits in controlled release profiles, cell permeability as well as fewer adverse effects (such as hand - foot syndrome and hypersensitivity reaction) (Nishiyama and Kataoka, 2006). Furthermore, Polymeric nanomicelles possess several prominent advantages over conventional surfactant micelles in that they have better thermodynamic stability in physiological solution, as indicated by their low critical micellar concentration (CMC), which makes the nanomicellar formulation stable and prevents their rapid dissociation in vivo (Muthu et al., 2009).

Currently, several passive targeted nanomicelles containing anticancer drugs, such as paclitaxel (extracted from the needles of yew trees Taxus baccata L.), doxorubicin and cisplatin, are already under preclinical and clinical invetigations (Nishiyama and Kataoka, 2009). Pharmacokinetic and bioavailability studies that have been conducted for these pharmaceuticals of great practical importance are critically evaluated. Xiao and co-workers labeled a micelle system covalently with ¹²⁵I and loaded the nanomicelles with ¹⁴C-paclitaxel. The liquid scintillation counting confirmed that ¹⁴C-labeled paclitaxel sequestered in nanomicelles had increased uptake by tumor tissue and slower pharmacokinetics than Taxol^® and may be clinically useful for both tumor imaging and improved chemotherapy applications (Xiao et al., 2012). Generally speaking, nanomicelles, from a pharmacokinetic point of view, could be exploited as promising highly efficient drug delivery vehicles for anti-tumor drugs.

Till date, neither extensive in vivo nor in vitro investigations have been reported by any other researchers on the effectiveness of a mixed micellar nanoformulation as a delivery system to enhance oral bioavailability of FVS. In this study, anti-tumor components were initially screened from F. velutipes using lipid rafts chromatography based on tyrosine kinase receptor (unpublished results). This was followed by methyl thiazolyl tetrazolium (MTT) bioassay of the isolated compounds and then the preparation of the nanomicelles formulation (a polyphase dispersed system containing PVP-phospholipid-sodium cholate loaded with FVS). The F. velutipes sterol nanomicelles (FVSNs) were finally evaluated for its bioavailability and selective tissue distribution as against the free FVS using rats and mice respectively.