Design and ocular tolerance of flurbiprofen loaded ultrasound-engineered NLC

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Abstract

Packaging small drug molecules, such as non-steroidal anti-inflammatory drugs (NSAIDs) into nanoparticulate systems has been reported as a promising approach to improve the drug's bioavailability, biocompatibility and safety profiles. In the last 20 years, lipid nanoparticles (lipid dispersions) entered the nanoparticulate library as novel carrier systems due to their great potential as an alternative to other systems such as polymeric nanoparticles and liposomes for several administration routes. For ocular instillation nanoparticulate carriers are required to have a low mean particle size, with the lowest polydispersity as possible. The purpose of this work was to study the combined influence of 2-level, 4-factor variables on the formulation of flurbiprofen (FB), a lipophilic NSAID, in lipid carriers currently named as nanostructured lipid carriers (NLC). NLC were produced with stearic acid (SA) and castor oil (CO) stabilized by Tween® 80 (non-ionic surfactant) in aqueous dispersion. A 24 full factorial design based on 4 independent variables was used to plan the experiments, namely, the percentage of SA with regard to the total lipid, the FB concentration, the stabilizer concentration, and the storage conditions (i.e., storage temperature). The effects of these parameters on the mean particle size, polydispersity index (PI) and zeta potential (ZP) were investigated as dependent variables. The optimization process was achieved and the best formulation corresponded to the NLC formulation composed of 0.05 (wt%) FB, 1.6 (wt%) Tween® 80 and a 50:50 ratio of SA to CO, with an average diameter of 288 nm, PI 0.245 of and ZP of −29 mV. This factorial design study has proven to be a useful tool in optimizing FB-loaded NLC formulations. Stability of the optimized NLC was predicted using a TurbiScanLab® and the ocular tolerance was assessed in vitro and in vivo by the Eytex® and Draize test, respectively. The developed systems were shown physico-chemically stable with high tolerance for eye instillation.

Introduction

One of the most common disorders in ophthalmic therapy is the ocular inflammatory disease affecting any part of the eye or the surrounding tissues [1], [2]. Inflammation involving the eye can range from the familiar allergic conjunctivitis of hay fever to rare [3], potentially blinding, conditions such as keratitis, scleritis or episcleritis, uveitis, optic neuritis, orbital pseudo-tumour and chronic conjunctivitis [1]. Others, e.g. the postoperative inflammation [4], are characterized by a severe inflammation usually affecting the uvea. Since uvea presents many blood vessels nourishing various parts of the eye, this structure plays an important role in the human vision mechanism. Thus, the presence of a damaging inflammatory disorder may lead to vision impairment [5].

Topically applied non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in the management and prevention of ocular inflammation and cystoid macular edema (CME) related to cataract surgery and the maintenance of mydriasis during cataract surgery [6], [7]. Although steroidal agents have been the standard treatment for ocular inflammation [8], [9], the use of NSAIDs has increased over the past two decades [8], [10], [11], [12]. The main advantage of using topical NSAIDs is the avoidance of undesirable effects of steroidal agents, namely, the decreased immunological response to infection, cataract formation, steroid-induced raised intraocular pressure (IOP) and inhibition of re-epithelisation following epithelial denudation [13]. However, several NSAIDs have been associated with some side effects, mainly affecting the gastrointestinal tract [14].

Flurbiprofen (FB), a water insoluble and acidic anti-inflammatory drug, is currently used as a first line ophthalmic medication for the inhibition of miosis induced during the course of cataract surgery [15]. In order to minimize occurrence of miosis and to improve therapeutic efficiency of NSAIDs, the development of novel delivery systems for ocular instillation is a demand. Within these approaches, nanostructured lipid carriers (NLC) have been gathering attention from researchers worldwide [16], [17]. NLC are systems composed of a solid lipid matrix with a certain content of liquid lipid useful to increase the solubility of lipophilic drugs. These carriers show great promise as drug delivery devices for the eye, due to their biocompatibility, modified release kinetics [18], avoidance of organic solvents during the production process, easy large scale production and reduction of drug leakage during storage. Different techniques can be employed to prepare lipid carriers, being the hot homogenization technique the most commonly applied [19]. Nevertheless, it requires the use of appropriate devices which are not commonly available in research labs. On the other hand, ultrasounds are frequently used to disperse two immiscible phases, such as lipid and water, and can be easily applied to produce nanosuspensions of lipid materials [20], [21]. This technology is based on the extreme conditions generated within the collapsing cavitational bubbles of the inner phase leading to size reduction [22]. Ultrasonic processing is fast and highly reproducible if the operating parameters (e.g., ultrasonication time and power, operating temperature), are critically controlled. Ultrasound probes are practically self-cleaning, they account for negligible sample losses, and can be used for high scale production. The factorial design is frequently used for the planning of a research because it provides the maximum amount of information and requires the least amount of experiments [23]. Although the common method of experimental design to optimize operating conditions is by changing 1 variable at a time, this is time-consuming and gives no guarantee for optimal parameter determination.

This paper reports a factorial design approach as a guideline for the development and characterization of a new ocular delivery system, namely FB-loaded NLC, composed of stearic acid (SA) and castor oil (CO). FB-loaded NLC were produced by an ultrasound method and the tolerance of the optimized formulation was evaluated by the Eytex® in vitro test and by the Draize in vivo test. In order to detect destabilization phenomena stability studies were also undertaken.

Section snippets

Materials

Flurbiprofen and Tween® 80 (polyoxyethylene sorbitan monooleate) were purchased from Sigma Aldrich (Madrid, Spain). Lipids investigated included stearic acid (a saturated fatty acid (of C18) provided by Croda Industrial Specialities (Nettetal, Germany), Precifac® ATO (cetyl palmitate), Compritol® ATO 888 (glyceryl behenate), Precirol® ATO 5 (glycerol mono, di and tripalmitostearate) and Precirol® ATO WL2155 (glyceryl ditristearate) gifted by Gattefossé (Gennevilliers, France), Dynasan® 116

Results and discussion

The first step in formulating NLC dispersions is always the assessment of drug solubility in the lipid phase. This was performed by dissolving increasing concentrations of FB in various solid and liquid lipids, previously selected on the basis of their suitability for ocular administration [36]. To check the drug solubility in the solid lipids, mixtures were melted at a temperature approximately 10 °C above the melting point of the solid lipid, and the maximum FB concentration that could be

Conclusions

This study reports a first approach on the use of a 2-level 4-factors factorial design in the optimization NLC formulations produced by an ultrasound method for the encapsulation of flurbiprofen (a lipophilic NSAID drug). The derived polynomial equations and Pareto Charts proved to be satisfactory in predicting the dependent variable values for the preparation of optimum NLC with desired mean particle size, polydispersity index and surface electrical charge for ocular instillation. Optimal

Acknowledgements

This work was supported by the Ministry of Science and Innovation of the Spanish Government (R&D and Innovation Project CTQ2005-09063-C03-03) and by the Spanish-Portuguese Integrated Actions research program (HP2008-0015).

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