Preparation and Evaluation of Methoxypolyethylene Glycol-Poly (DL-Lactic Acid) Nanoparticles Loaded with Glycyrrhetic Acid

Research Article

Austin J Nanomed Nanotechnol. 2016; 4(1): 1043.

Preparation and Evaluation of Methoxypolyethylene Glycol-Poly (DL-Lactic Acid) Nanoparticles Loaded with Glycyrrhetic Acid

Hiraku Onishi*, Ken-ichiro A, Sasatsu M, Ikeuchi Y and Machida Y

Department of Drug Delivery Research, Hoshi University, Japan

*Corresponding author: Hiraku Onishi, Department of Drug Delivery Research, Hoshi University, 2-4-41, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan

Received: March 22, 2016; Accepted: May 30, 2016; Published: June 02, 2016

Abstract

Glycyrrhetic acid (GLA)-loaded methoxypolyethylene glycol-poly (DL-lactic acid) (PLA-MPEG) nanoparticles, named NP, were produced and evaluated in vitro and in vivo. NP were prepared by a simple O/W emulsification/evaporation method using 1% PVA as a surfactant. NP with the size of 300 – 400 nm and more than 3 % GLA content were obtained at the PLA-MPEG/GLA ratio of 6/1 (w/w). The NP were examined for in vitro release in PBS (pH 7.4). NP exhibited an initial burst of approximately 45 % and released 30 % slowly during 7 d. Furthermore, NP and GLA alone were examined for efficacy using mice with carbon tetrachloride-induced hepatitis. NP tended to show better and longer hepato-protective effect than GLA alone. NP are suggested as a potential delivery system of GLA for the liver injury.

Keywords: Glycyrrhetic acid; Methoxypolyethylene glycol-poly(DL-lactic acid); Nanoparticles; In vitro release; Hepato-protective effect

Introduction

Licorice root is a widely-used traditional medicine, and its major ingredient is glycyrrhizin (GLZ) [1,2]. GLZ exhibits various useful biological functions such as anti-allergy [3], anti-inflammation [4], anti-hepatitis [1,2,5], anti-virus [6,7] and interferon-γ inducing effect [1,8]. However, GLZ causes toxic side effects like edema and hypertension by its corticoid-like action called pseudo-aldosteronism [1]. Glycyrrhetic acid (GLA) is produced by intestinal bacteria after oral ingestion of GLZ [9]. GLA also has various biological functions containing anti-inflammatory functions [1,10,11]. In particular, GLA is more highly hepato-protective than GLZ [1,5]. As GLA also induces side effect like pseudo-aldosteronism, its use is often limited. Therefore, it is desirable to suppress its dosing amount and administration frequency.

Micro- and nanoparticulate dosage forms have been used as drug delivery techniques to improve the drug action. Since poly (DLlactic acid) (PLA), poly (DL-lactic acid-co-glycolic acid) (PLGA), methoxypolyethylene glycol (MPEG)-PLA block copolymer (PLAMPEG) and MPEG-PLGA block copolymer (PLGA-MPEG) are safe and easy to process, their microparticles and nanoparticles have been produced as drug delivery devices [12-15]. These microand nanoparticles can be used parenterally due to their highly safe properties. Previously, PLGA-based microparticles containing GLA (PLGA/GLA-M) were produced and examined for the hepato-protective function [16,17]. PLGA/GLA-M exhibited good localization to liver and prolonged release there, resulting in better hepato-protective effect than GLA alone.

However, PLGA/GLA-M needed to be washed with the mixture of methanol and phosphate-buffered saline (PBS) of pH 7.4 twice or more; because non-washed or water-washed PLGA/GLA-M showed a high initial rapid release of nearly 80 %. On PLA-MPEG and PLGAResearch MPEG, composed of hydrophobic and hydrophilic polymer chains, have been found to easily form nanoparticles in aqueous media. In addition, as those nanoparticles are highly safe and disperse well in aqueous media, they have been used as drug carriers for parenteral drug delivery systems. In this study, the development of PLA-MPEG nanoparticles containing GLA, called NP, has been attempted, and the obtained NP have been evaluated from the in vitro features and hepato-protective effectiveness in vivo using rats with carbon tetrachloride (CCl4)-induced hepatitis.

Materials and Methods

Materials

DL-Lactide was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Stannous octoate (St-Oct), methoxypolyethylene glycol (MPEG; MW 2,000) and 18β-glycyrrhetic acid (GLA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of reagent grade.

Animals

Six-week old male ddY mice were purchased from Tokyo Laboratory Animal Science Co. Ltd. (Tokyo, Japan). They were housed with the breeding diet MF produced by Oriental Yeast (Tokyo, Japan) and water under the room conditions of 23 ± 1 oC and 60 ± 5 % relative humidity. The animal experimental protocol was approved Animal Research Committee of Hoshi University and the experiments were performed according to the Guideline Principles of Animal Care and Use of Hoshi University.

Preparation of nanoparticles

Methoxypolyethylene glycol-poly(DL-lactic acid) block copolymer (PLA-MPEG) was synthesized by referring to the methods by Gref et al. [14] and Bazile et al. [15], namely, a ring opening polymerization using St-Oct as a catalytic agent and subsequent purification by repeatedly putting the polymer-containing dichloromethane solution into water. The chemical structure of the product was examined using AV-400M digital NMR (Bruker BioSpin K.K., Yokohama, Japan). The polymerization degree, substitution degree of MPEG and molecular weight of PLA-MPEG were examined in the same manner as had been reported before [18,19]; namely, they were calculated by comparing the integrated intensity of each proton signal with that of the proton signal of the PLA terminal methine [18,19].

PLA-MPEG nanoparticles loaded with GLA, named NP, were produced as follows. PLA-MPEG (30 mg) and GLA (2.5, 5 and 10 mg) were dissolved in 1 mL of dichloromethane, and the solution was put into 0.01% HCl aqueous solution containing 1% (w/v) PVA. The mixture was stirred with a vortex mixer for 30 s, and sonicated at 28 Hz (100 W) for 30 min. The resultant emulsion was stirred with a magnetic stirrer at 25 oC for 2 h. The obtained suspension was condensed by evaporation in vacuo, and underwent gel-filtration using a Sephadex G50 column using water as elution solvent. The high molecular weight fractions were obtained as aqueous suspension of nanoparticles (NP).

Measurement of particle size and drug content

The particle size of NP was determined by measuring the dynamic light scattering of NP suspension with an ELS-800 apparatus (Otsuka Electronic Co., Ltd., Osaka, Japan). The drug content of NP was measured as follows. Acetonitrile (1.5 mL) and HPLC mobile (0.5 mL) were added to 0.1 mL of NP aqueous suspension, and the resultant solution was analyzed by HPLC on the concentration of GLA.

The NP amount in their aqueous suspension was determined as follows. After the NP aqueous suspension (3 mL) was freeze-dried, the residue and p-hydroxybenzoic acid (1 mg) were mixed in CDCl3, and the 1H-NMR spectrum of the resultant solution was measured. The integrated intensity of the proton signal of the PLA terminal methine was compared with that of the proton signal of p-hydroxybenzoic acid, leading to the amount of PLA-MPEG in the NP suspension. The GLA content was calculated from the ratio of (GLA amount)/ (NP amount).

Release of GLA from nanoparticles in PBS

The NP aqueous suspension was diluted 10 times with PBS of pH 7.4. The resultant suspension was divided into test tubes with equal volume of 2 mL. Each sample was incubated at 37oC. At 3 h, 7 h, 1 d, 2 d, 4 d and 7 d after the start of the incubation, three tubes were taken and centrifuged at 40,000 rpm for 20 min. After the supernatant was mixed with the same volume of HPLC mobile phase, the resultant solution was examined by HPLC for the concentration GLA, which gave the release amount of GLA.

The release profiles were further examined in the presence of rat plasma. Namely, the NP aqueous suspension was diluted 10 times with the mixture of murine fresh plasma and PBS (pH 7.4), and incubated in a manner similar to that stated above. At 2 h, 7 h and 24 h, the NP was precipitated similarly, and the supernatant was taken. Dichloromethane (5 mL) and 0.1 M acetate buffer of pH 5 (1 mL) were added to the supernatant (0.1 mL), and the mixture was shaken vigorously and centrifuged. The organic phase (4 mL) was taken and evaporated, and the resultant residue was dissolved in methanol (200 μL). The resultant solution was analyzed by HPLC, in which the data were corrected by the extraction ratio [16].

Therapeutic effect in mice with carbon tetrachlorideinduced hepatitis

The in vivo studies were conducted as shown in Figure 1, in which therapeutic effects of NP and GLA alone were tested at single and twice administration schedules. First, hepatitis model mice were produced by i.p. injection of a 25 % (v/v) carbon tetrachloride (CCl4) solution in olive oil at 4 mL/kg to each mouse at 0 and 48 h. NP suspension or GLA suspension in 2 % (w/w) Tween 20 aqueous solution, called GLA alone, was administered intravenously via tail vein at 5 mg/10 mL/kg at 3 h after the first CCl4 injection (single administration), and at 3 and 27 h after the first CCl4 injection (twice administration). The blood sampling was performed just before and at 24, 48, 72 and 96 h after the first CCl4 injection. Just after each sampling, the plasma was obtained by centrifugation