Effect of sterilization on the physical stability of brimonidine-loaded solid lipid nanoparticles and nanostructured lipid carriers

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Abstract

Nanoparticulate delivery systems have recently been under consideration for topical ophthalmic drug delivery. Brimonidine base-loaded solid lipid nanoparticles and nanostructured lipid carrier formulations were prepared using glyceryl monostearate as solid lipid and were evaluated for their physical stability following sterilization by autoclaving at 121 °C for 15 min. The objective of this work was to evaluate the effect of autoclaving on the physical appearance, particle size, polydispersity index, zeta potential, entrapment efficiency and particle morphology of the prepared formulations, compared to non-autoclaved ones. Results showed that, autoclaving at 121 °C for 15 min allowed the production of physically stable formulations in nanometric range, below 500 nm suitable for ophthalmic application. Moreover, the autoclaved samples appeared to be superior to non-autoclaved ones, due to their increased zeta potential values, indicating a better physical stability. As well as, increased amount of brimonidine base entrapped in the tested formulations.

Introduction

Nanoparticulate delivery systems have recently been under consideration for topical ophthalmic drug delivery. They can incorporate lipophilic, hydrophilic and poorly water soluble drugs. The low toxicity profile of this delivery system permits repeated administration of therapeutic agents (Attama et al., 2007). The lipids are solid both at body and room temperatures. The solid matrix of solid lipid nanoparticles (SLNs) tightly bind the drug molecules, hence reducing the mobility of incorporated drugs compared to conventional liquid colloidal drug delivery systems, allowing controlled drug release. Also, protection of sensitive drugs from degradation was achieved by incorporating drugs in the solid lipid matrix (Müller et al., 1995). Moreover, SLNs have proven to prolong the retention of drugs on the ocular surface and subsequently improve their corneal penetration (Cavalli et al., 2002) and enhance ocular bioavailability due to their lipophilic character (Attama et al., 2008).

SLNs can be formulated as stable nanodispersions in liquid dosage forms. Therefore, they can be administered as eye drops based on colloidal suspension. Such a formulation avoids blurred vision and is comfortable due to the small SLNs size (Gokce et al., 2008). The crystallinity of solid lipids affects the release properties of the SLNs. Immediately after preparation, lipids partially crystallize in high energy modifications with many imperfections in the crystal lattice (Müller et al., 2007). During storage, these modifications can transform into low energy modifications. Transformation into a highly ordered crystal structure, reduces the number of imperfections in the crystal lattice, leading to drug expulsion (Müller et al., 2002a, Gonzalez-Mira et al., 2011). To overcome limitation attributed to the high SLNs matrix crystallinity, nanostructured lipid carriers (NLCs) were produced as a second generation derived from SLNs, exhibiting many excellent features for application as a drug carrier e.g. controlled release of incorporated drugs, increased loading capacity, in addition to improved physical and chemical long term stability (Müller et al., 2002a, Müller et al., 2002b, Tian et al., 2012).

In this study, brimonidine; was chosen as a model drug; it is a highly selective α2-adrenergic agonist (Acheampong et al., 2002). Clinical trials have proved that, topical brimonidine tartrate is safe and effective to lower the intraocular pressure (IOP) in patients with open angle glaucoma or ocular hypertension as an alternative or adjunct to topical β-blocker therapy (Cantor et al., 2008, Noecker, 2006).

It reduces IOP by a dual mechanism of action; reducing aqueous humor production and increasing aqueous humor outflow via the uveoscleral pathway (Dipti et al., 2009, Biner et al., 2001).

Ophthalmic products usually have a short shelf-life and they must be used within 1 month after opening due to contamination risks. Sterilization is a required step for all ophthalmic preparations it should not change the formulation properties; regarding the physical and chemical stability and the drug release kinetics (Mehnert and Mäder, 2001). A sterilization technique has to be carefully chosen to ensure formulation sterility without degradation or aggregation of the solid lipids to avoid toxicity and instability (Mehnert and Mäder, 2001).

Commonly used techniques for sterilization are γ-radiation, filtration, aseptic production and autoclaving. Free radicals are formed after sterilization by γ-radiation due to the high energy of these rays (Mehnert and Mäder, 2001). The free radicals can be either returned to original molecules without chemical modification or cause chemical instability and degradation, depending on the reactivity of the sample component. In addition, high molecular mobility (e.g., liquid or semisolid formulations) and the presence of oxygen favors γ-sterilization-induced degradation reactions. Thus, chemical changes of the lipid components were reported following sterilization by γ-radiation (Sculier et al., 1986). Previous studies showed that; γ-irradiation could be an alternative method to steam sterilization for temperature sensitive samples (Schwarz and Mehnert, 1995, Schwarz et al., 1995).

Filtration technique as well as, aseptic preparation strategy can be also employed for SLNs sterilization, similar to the sterilization of parenteral emulsions and nutrition (Muéller et al., 2000). However, filtration sterilization of dispersed systems requires high pressure and is not applicable to particle sizes (PS) above 0.2 μm (Mehnert and Mäder, 2001). Production under aseptic conditions is possible, but it is very complex and expensive (Heiati et al., 1998).

One of the advantages of SLNs over other colloidal systems is that they can be sterilized by autoclaving, a commonly used and reliable technique. During autoclaving SLNs melt and recrystallize in a controlled manner. Thus, certain structural features assigned to SLNs through controlling the production parameters will be lost by autoclaving, the severity of which is defined by the composition of the SLNs (Muéller et al., 2000).

However, it should be noted that heating can induce physical instability and particle aggregation. An increase in the average PS is usually observed after sterilization by heating. In addition, an important issue which should be considered is; the degradation of lipids during sterilization which can cause potential toxicity (Mehnert and Mäder, 2001).

It should be kept in mind that degradation does not always cause increased PS. In contrast, the formation of species like lysophosphatides or free fatty acids might even preserve small PS, but might cause toxicological problems (Mehnert and Mäder, 2001).

Critical parameters affect SLNs stability including; sterilization temperature and SLNs composition. The correct choice of the emulsifier is significantly important for the physical stability of the sample at high temperatures. Increased temperatures, affect both the mobility and the hydrophilicity of all emulsifiers to different extents. Steam sterilization causes the formation of an o/w emulsion due to the melting of the lipid particles, then solid particles are formed after re-crystallization (Mehnert and Mäder, 2001).

In the present the effect of autoclaving at 121 °C for 15 min on the physical stability of two SLNs and NLCs formulations; was studied. In addition, the effect of autoclaving on the rheological properties of the prepared formulations and the thermal properties of the bulk lipid glyceryl monostearate (GMS) was investigated. Also, in-vivo ocular irritancy will be evaluated to ensure good ocular tolerance after SLNs autoclaving.

Section snippets

Materials

Brimonidine tartrate was kindly gifted by Jamjoom Pharmaceuticals, Jeddah, Saudi Arabia. Glyceryl monostearate and Kolliphor® P 188 (Pluronic F68) were kindly gifted by BASF Italia, Cesano Maderno, Italy. Castor oil, Morgan specialty chemicals, Cairo, Egypt. Visking® dialysis membrane, 36/32, 24 mm, molecular weight cut off (MWCO); 12,000–14,000; Serva, Heidelberg, Germany. Ethanol  99% purity was purchased from ADWIC, El-Nasr Pharmaceutical Chemicals, Co., Cairo, Egypt.

Precipitation of Brimonidine base from Brimonidine tartrate

A solution of brimonidine tartrate was treated with dilute ammonia (10% w/w) to precipitate a yellow solid of brimonidine base. This solid was filtered using a sintered glass filter by suction using a water pump, washed several times with distilled water to get rid of any remaining ammonia and air dried. The identity of the obtained Brimonidine base was confirmed by determining its melting point (m.p.), infra-red (IR) and proton nuclear magnetic resonance (1HNMR) spectra. The m.p. was

Clarity and physical appearance

The clarity of the nanodispersions (SLNs and NLCs) was determined by visual examination under light before and after sterilization.

Particle size and polydispersity index

The PS and polydispersity index (PI) of the autoclaved and non-autoclaved brimonidine base-loaded SLNs and NLCs dispersions were determined through dynamic light scattering technique at 25 °C (Zetasizer, NanoZS, Malvern, UK). (Luykx et al., 2008). The samples were suitably diluted with 0.45 μm filtered distilled water before measurement. Samples were measured in

Results and discussion

Brimonidine tartrate is soluble in water (34 mg/ml) (Ali et al., 2009). Preliminary studies were done to prepare SLNs encapsulating the hydrophilic brimonidine tartrate using the cold high shear homogenization method but all trials resulted in a very low entrapment of brimonidine tartrate into the solid lipid. Brimonidine tartrate 0.2% is equivalent to 0.132% w/w Brimonidine base. Thus, brimonidine tartrate salt was precipitated into the base which is poorly water soluble and thus, may allow

Conclusion

Both the prepared brimonidine base-loaded SLNs and NLCs formulations; were found to be physically stable after autoclaving at 121 °C for 15 min. This sterilization process yielded particles which are in nanometric range below 500 nm, suitable for ophthalmic application. Autoclaved samples appeared to be superior to non-autoclaved ones, due to the increased ZP values following autoclaving, indicating a better physical stability. As well as increased amount of brimonidine base entrapped in both SLNs

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