Development and optimization of ezetimibe nanoparticles with improved antihyperlipidemic activity

https://doi.org/10.1016/j.jddst.2018.12.001Get rights and content

Abstract

The current study aims to develop and optimize ezetimibe chitosan nanoparticles by using 22.31 factorial design, where chitosan: tripolyphosphate (TPP) ratio (X1), molecular weight of chitosan (X2) and drug concentration (X3) were chosen as independent variables. While, entrapment efficiency (Y1), drug loading capacity (Y2), particle size (Y3) and polydespersity (Y4) were selected as dependent variables. The optimized nanoparticles formula was F3 composed of low molecular weight chitosan at a ratio to TPP of 1.75: 2 and 30 mg drug. F3 optimizied nanoparticles were spherical with regular form as shown by TEM images. It was further evaluated for in vitro release and in vivo antihyperlipidemic activity in comparison with the marketed product (choletimibe). The hyperlipidemic rats treated with optimized formula showed significantly more superior reducing effect on lipid profile parameters and improved atherogenic index compared with marketed product. The degree of this reduction was detected by the following percent of variations 29.62% for serum total cholesterol, 33.17% for triglycerides, 51.78% for LDL-C, 33.19% for VLDL-C, 43.16% for atherogenic index and 29.07% for total lipids as compared to marketed product. Based on our findings, the convenience and efficiency of the antihyperlipidemic activity of ezetimibe nanoparticles were well demonstrated.

Introduction

Abnormalities of lipid metabolism are one of the key factors, representing around 50% of the population-attributable risk of developing cardiovascular disease [1]. Hyperlipidemia is a serious public health problem both in developed and developing countries where, it is considered as one of the five leading causes of death in the world [2,3]. It comprises a heterogeneous group of disorders of one or more lipid (cholesterol, cholesterol esters, phospholipids or triglycerides) and/or lipoprotein macromolecules (LDL-C and apolipoprotein B) and/or anti-atherogenic HDL-C in the blood circulation [4,5].

Ezetimibe (synthetic 2-azetidinone) is a lipid lowering agent used as a mono therapy or in combination with statins for the treatment of primary hypercholesterolemia that, characterized by good tolerance and low incidence of adverse effects. It selectively inhibits the intestinal absorption of dietary and biliary cholesterol without affecting the absorption of fat-soluble nutrients. EZE (Fig. 1), is categorized as BCS Class II compound (poorly water soluble and highly permeable) with a relative bioavailability range from 35 to 65%, it inhibits the cholesterol absorption from the small intestine by blocking the Niemann-Pick C1 like l protein [6]. The variable bioavailability of EZE may be due to its very low solubility and dissolution rate, in addition to extensive efflux by p-glycoprotein (P-Gp) [7].

Nanotechnology is the engineering of functional systems with an appropriate particle size with narrow size range, increased dissolution rate as a result particle size reduction which in turn can enhance the bioavailability. Nanosystems such as nanoparticles (NPs), micelles and liposomes are of the most progressive strategies for the delivery of either hydrophilic or hydrophobic active compounds, depending on the carrier nature [[8], [9], [10], [11]]. Nanoparticles (NPs) multifunctionality and wide range of chemical, physical or biological properties have received a considerable attention as an innovative and promising carrier able to overcome many of the undesirable drug properties while maintaining or improving its therapeutic efficacy [[12], [13], [14]]. Besides, NPs could enhance drug stability, sustaining drug release, targeting to infected tissue, prolonging drug effect in the target tissue and targeting drug effect upon-inclusion of specific ligands [[15], [16], [17]].

Nanoparticles based on polysaccharides are revolutionizing medical treatments because of their high effectiveness and suitability for drug delivery and for the design of personalized therapies. Chitosan is a natural cationic linear heteropolysaccharide composed of units of N-acetylglucosamine and glucosamine, linked by −1,4 bonds and showed multiple properties such as biocompatibility, biodegradability, bioadhesion, absence of allergenicity and toxicity. Therefore, chitosan and its derivatives have been used to encapsulate drugs for the pharmaceutical and biomedical fields owing to its excellent biocompatibility, nontoxicity, mucoadhesive properties and good drug permeability [[18], [19], [20]].

Chitosan nanoparticles could be prepared by ionic gelation, which is a simple and safe method that has been widely used, based on the transition of the polymer from liquid state to a gel. CS-TPP complex was formed by the ionic interaction between positively charged CS and negatively charged phosphoric ions of TPP.

The aim of this study was to maximize entrapment efficiency (EE) and drug loading capacity (LC) and minimize particle size (PS) and polydispersity index (PDI) of EZE nanoparticles by the optimization of the effect of molecular weight of chitosan, ratio of chitosan: TPP and drug concentration to achieve improved antihyperlepidimeic activity for ezetimebe loaded CS/TPP nanoparticles. Moreover the in vitro drug release was evaluated in order to elucidate the ability of the optimized formulation to release ezetimibe.

Low molecular weight chitosan (LMWt, 50,000–190,000 Da), High molecular weight chitosan (HMWt, 310,000–375,000Da), acetic acid, tween 80 and tripolyphosphate (TPP) were purchased from Sigma Aldrich Co. (USA). Choletimb and Ezetimibe was kindly gifted from Marcyrl International Company (Egypt). Acetonitrile (HPLC grade), disodium hydrogen phosphate and potassium dihydrogen phosphate were purchased from Sigma Aldrich Co. (USA). Cholesterol and cholic acid were purchased from Sigma Aldrich chemicals (USA). In vitro diagnostic kits for determination of cholesterol, triglycerides, high density lipoprotein cholesterol (HDL-C) and total lipids were purchased from Biodiagnostic (Cairo, Egypt).

Chitosan nanoparticles loaded with EZE were prepared by ionotropic gelation process. The mechanism of ionic gelation is that the interaction between the negatively charged polyanion sodium tripolyphosphate (TPP) and the positively charged amino groups of chitosan [21,22]. Two Chitosan solutions (LMWt chitosan and HMWt chitosan) were prepared in 1% acetic acid. TPP was dissolved in water. Both chitosan and TPP solutions have been stirred overnight using magnetic stirrer till complete dissolution. After that, chitosan solution was added to ezetimibe and tween 80 (0.5%) with continuous stirring. Tween 80 (a nonionic surfactant), was added to increase solubility and as a stabilizer to prevent the agglomeration of nanoparticles. Nanoparticles were obtained upon the drop wise addition of TPP aqueous solution to a chitosan solution (containing EZE and tween 80) under homogenizer at 4000 rpm and stirring was maintained for 15 min at room temperature. Nanoparticles were collected by centrifugation at 10,000 rpm for 1 h and supernatants were discarded.

Section snippets

Entrapment efficiency (EE) and loading capacity (LC)

The percentage of EZE loaded in nanoparticles was determined, as the prepared nanoparticles were subjected to centrifugation at a speed of 10,000 rpm for 1 h and the free drug content in the supernatant was determined by validated HPLC method [23]. The used column was a Zorbax C18 column (4.6 × 250 mm, 5 μm particle size). Mobile phase was a mixture of acetonitrile: phosphate buffer pH 3.15 (42:58). Mobile phase flow rate was set at 0.8 ml/mn and UV detection wavelength was fixed at 230 nm.%EE=

Experimental animals

Adult male albino rats, weighing 220 ± 30 g obtained from the animal house of the National Organization for drug control and Research (NODCAR, Cairo, Egypt) were used in the present study. Rats were kept for two weeks of acclimatization and given food and water ad libitum and maintained under Good Laboratory Practices (GLP) at 23 ±2 °C, 12/12 h light/dark cycles and were housed in stainless steel cages (3 rats per cage). The experimental protocol was approved by the local animal care committee,

Results and discussion

In our design twelve formulations were suggested by the 22.31 factorial design for three independent variables: ratio of chitosan:TPP (X1) and molecular weight of chitosan (X2) at two levels and drug concentration (X3) at three levels. The effect of these factors on EE (Y1), LC (Y2), PS (Y3) and PDI (Y4) is presented in Table 2.

Conclusion

The present study showed the development and optimization of EZE chitosan nanoparticles to get a formula with the least PS and PDI associated with the highest EE and LC. A full 22.31 factorial design was used to determine the optimal formulation. The optimum formulation was F3 encompassing 30 mg EZE together with LMWt chitosan at 1.75:2 chitosan: TPP. It exhibited significant increase of drug release in phosphate buffer pH 6.8 compared to the marketed product. The optimized F3 chitosan

Declaration of interest

The authors report no conflicts of interest.

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