Preparation and in vitro evaluation of apigenin-loaded polymeric micelles

https://doi.org/10.1016/j.colsurfa.2013.03.051Get rights and content

Highlights

  • A novel apigenin-loaded mixed micelle was prepared and optimized.

  • The solubility of apigenin in the micelles was 148 times that of crude apigenin.

  • CMC of the mixed micelles was as low as 4.23 × 10−5 mol/L.

  • The cytotoxicity against HepG2 and MCF-7 cells shows enhanced inhibition rate.

Abstract

The objective of this study was to prepare apigenin-loaded polymeric micelles to improve the solubility of apigenin in water. The polymeric micelles composed of Pluronic P123 and Solutol HS 15 were prepared by a thin-film dispersion method and the formulation was optimized with a central composite design (CCD). The obtained micelles were spherical under transmission electron microscope (TEM) with an average diameter of 16.9 nm, the entrapment efficiency and drug loading was 96.36% and 1.32%, respectively. The in vitro drug release study showed nearly 84% of apigenin was released from micelles within 36 h, showing the sustained release property. Cell toxicity assay indicated that the cytotoxicity of apigenin-loaded polymeric micelles against HepG2 and MCF-7 cancer cell in vitro was remarkably higher than that of the free drug. All the results provided the evidence for the great potential of polymeric micelles as nanocarrier for apigenin as a poorly water soluble drug.

Introduction

Flavonoids are a group of phenolic plant pigments and common constituents of human diet as well, presenting in most fruit and vegetables [1]. They are considered as dietary for a function of anti-cancer and anti-oxidant [2]. In several cases, flavonoids have been reported to possess anti-cancer potential [3], [4]. Apigenin (4′,5,7-trihydroxy-flavone) as a common flavonoid is believed to possess the preventive and therapeutic potential against cancers. It has been shown to possess anti-inflammatory, free radical scavenging properties with superiority of non-mutagenic and low toxicity [5]. More importantly, according to previous studies, apigenin could induce apoptosis of cancer cells and exert effects on inhibiting the invasive process [6]. However, the solubility of apigenin as a poorly water soluble drug is only 2.16 μg/mL in water [7] and 0.001–1.63 mg/mL in high hydrophilic or nonpolar solvents [8], leading to a poor absorption in gastrointestinal tract. Therefore, the improvement in solubility and bioavailability is urgently needed for development and application of apigenin.

Over the past two decades, nanoscale micelles have demonstrated great potential in delivering anticancer drugs [9], [10]. Polymeric micelle has a particle size ranging from 10 to 100 nm, preventing from being recognized by reticuloendothelial system (RES), increasing the systemic circulation time. On the other hand, the small size (<100 nm) of micelles allows for their efficient accumulation in tumor tissues via the enhanced permeability and retention (EPR) effect. Moreover, polymeric micelle has many other special properties such as biocompatibility and increased stability. Conventionally, this kind of micelle was composed of amphiphilic block copolymers. The hydrophobic part may face to face to form a hydrophobic core while the hydrophilic chains crosslinking outside forms a shell when the block copolymer self-assembles in water [11]. Consequently, the hydrophobic drugs can easily be encapsulated into the hydrophobic cores of the polymeric micelles.

P123 is one of the most common representatives of amphiphilic block copolymers, whose structure is PEO–PPO–PEO [12]. The PPO part is hydrophobic, leading to an aggregation against water and providing a local hydrophobic microenvironment where the hydrophobic drug can be dissolved, while the hydrophilic PEO part maintains the dispersion stability of the formed micelles. In P123, the proportion of PPO part is as high as 70%, which means micelles formed by P123 should have sufficient capability for solubilizing the poorly water soluble drugs because of the large bulk volume of inner core. However, the proportion of PEO blocks in P123 is only 30%, forming a relative thin shell of the micelle with a poor dispersibility. In addition, almost 5.4% PEO blocks exist within the core of the micelles at a low temperature under 30 °C [13], which decreases the hydrophobicity of core as well as the hydrophility of shell of simple P123 micelles. The unbalanced ratio of the PEO and PPO chains in P123 results in the destabilization or unstability of the micelle made from this block copolymer [14]. Therefore, it is obliged to modify the deficiency of simple P123 micelles aiming for preferred micelle stability.

According to previous study, mixed micelles composed of two or more kinds of polymer manifest synergistic properties, such as increased micelle stability and more effective solubilization capacity, superior to those composed of the individual component [15]. Solutol HS15 (polyethylene glycol-660 hydroxystearate), consisting of polyglycol mono- and diesters of 12-hydroxystearic acid, which has been recorded in the European Pharmacopoeia, is recommended as non-ionic solubilizing agent to be added to injection solutions [16]. Particularly, nanoformulations made from Solutol HS15 may show a long circulating action for the presence of about 30% of free polyethylene glycol (PEG) in Solutol HS15 [17], PEG can increase stability of colloidal dispersion system and enhance blood circulation time by decreasing macrophage up-take and complement activation [18], [19]. The composition of Solutol HS15 can make it form mixed micelle combined with other copolymer, and improve the related properties of the micelle made from this copolymer. For example, Li et al. fabricated a propofol-loaded mixed micelles formed from mPEG-PLA and Solutol HS15, and found the solubilization of propofol by the mixed micelles was more efficient than those made of mPEG-PLA alone [16]. Ma et al. developed docetaxel-loaded mixed micelles whose main constituents were Solutol HS 15 and lipid S100. A satisfactory encapsulation efficiency (87.6% ± 3.0%) was achieved and the areas under the curve ((0–6 h)) levels of docetaxel in blood and tumors were significantly higher in the mixed micelle group (15.9 ± 3.2 μg/mL, 601.1 ± 194.5 μg/g) than that in the docetaxel injection group (7.2 ± 1.7 μg/mL, 357.8 ± 86.2 μg/g), which showed that the mixed micelles significantly improved the bioavailability [20]. Based on the reports, we hypothesize that mixed micelles made from P123 and Solutol HS15 may allow for the higher drug encapsulation, the better stability and higher anticancer efficiency.

In the study, an apigenin-loaded polymeric micelle system with P123 and Solutol HS15 as mixed polymer carrier was prepared by a thin-film dispersion method, and the preparation process was optimized with a central composite design (CCD). In addition, the physicochemical properties and in vitro cytotoxicity of the optimized drug-loaded micelles were investigated.

Section snippets

Materials

Apigenin was obtained from TianCao chemicals Co. (Hangzhou, Zhejiang), Pluronic F127, Pluronic F68, Pluronic P123 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Missouri, USA). Solutol HS 15 was provided by BASF Co. (Germany), PEG (2000)-PLA (2000) was brought from DaiGang Biological Engineering Co.Ltd (Jinan, Shandong). All other chemicals were of analytical purity and commercially available.

Preparation of apigenin-loaded polymeric micelles

The apigenin-loaded micelles were prepared with a thin-film dispersion method [21].

The optimization of micelle formulation

The CCD is often applied to develop the response surface methodology aiming to optimize formulations with few of factors n (2  n  6), and it requires fewer experimental runs than factorial designs, providing savings on time and resources [25]. Before using the design, an overall influence factors during assembly process were investigated to distinguish the impact degree of each factor on the DL%. As a result of the preliminary experiments, the micelles formed by P123 had a higher DL% (0.30%) than

Conclusion

In the present study, apigenin-loaded micelles were fabricated by a thin-film dispersion method. The mixed polymeric micelles, composed of P123 and Solutol HS15, exhibited higher EE% and DL% for apigenin. The average size and the Zeta potential of the apigenin-loaded mixed micelles were 16.9 nm and −5.87 mV, respectively. The low CMC (4.23 × 10−5 mol/L) of the micelles meaned that they could keep a stable micelle structure even in a diluted environment. The in vitro release behavior of apigenin from

Acknowledgement

This work was partly supported by the Natural Science Foundation of Shandong Province, China (no. ZR2011 HM026).

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    These authors contributed equally to the work.

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