Preparation and In Vivo Toxicity Study of Solid Lipid Microparticles as Carrier for Pulmonary Administration

Preparation and In Vivo Toxicity Study of Solid Lipid Microparticles as Carrier for Pulmonary Administration
Submitted: November 11, 2003; Accepted: March 10, 2004
Vanna Sanna,1 Nathalie Kirschvink,2 Pascal Gustin,2 Elisabetta Gavini,1 Isabelle Roland,3 Luc Delattre,3 and Brigitte Evrard3
aaps PharmSciTech
1Dipartimento di Scienze del Farmaco, University of Sassari, via Muroni 23/a, 07100 Sassari, Italy
2Department for Functional Sciences, Section Pharmacology, Pharmacotherapy & Toxicology, B?t. B41, Boulevard de Colonster 20, Faculty of Veterinary Medicine, University of Li?ge, 4000 Li?ge, Belgium
3Department Pharmacie Gal?nique et Magistrale, B?t. B36, Avenue de l'H?pital, University of Li?ge, 4000 Li?ge, Belgium
Correspondence to:
Elisabetta Gavini
Tel: +39 079 228752
Fax: +39 079 228733
Email: eligav@uniss.it
Abstract
The purpose of this research was to investigate the effects of processing conditions on the characteristics of solid lipid microparticles (SLM) with a potential application as carriers for pulmonary administration. Compritol (5.0% wt/wt) SLM dispersions were prepared by rotor-stator homogenization, at different surfactant concentrations and emulsification times. The SLM were characterized, in terms of morphology and size, after lyophilization and sterilization by autoclaving process. In vivo assessment was carried out in rats by intratracheal instillation of either placebo or SLM dispersion, and by bronchoalveolar lavage for cytological analysis. Mean particle size of 4 to 5 ?m was achieved using 0.3% and 0.4% (wt/wt) of emulsifier (Poloxamer 188) and emulsification times of 2 and 5 minutes. The particles showed spherical shape and smooth surface. The morphology of microparticles, the size, and the size distribution were not substantially modified after lyophilization and sterilization. Total cell counts showed no significant differences between placebo and SLM 0.5% or 2.5% groups. Regarding cytology, percentage of polymorphonuclear neutrophils and macrophages did not significantly differ between groups. These results suggest that a single intratracheal administration of the SLMs does not induce a significant inflammatory airway response in rats and that the SLMs might be a potential carrier for encapsulated drug via the pulmonary route.
Introduction
In recent years, biocompatible lipid micro- and nanoparticles have been reported as potential drug carrier systems as alternative materials to polymers.1-3 Solid lipid particles combine several advantages and avoid the disadvantages of other colloidal carriers. The following are positive features of the potential use of solid lipid particles as drug carrier systems:
They offer the possibility of controlled drug release and drug targeting.4
They provide protection of incorporated active compounds against degradation.
Their solid matrix is composed of physiological and well-tolerated lipids.
They allow for hydrophilic and/or hydrophobic drugs to be incorporated.5,6
The drug solubility and miscibility in melted lipid, chemical and physical structure of lipid materials, and their polymorphic state determine the loading capacity of drug in the lipid particles.7 The amount of drug encapsulated can vary from 1% to 5% for hydrophilic compounds8,9 and up to 80% for lipophilic compounds.5,10
Solid microparticles in dispersions are usually obtained using a melt dispersion method or a solvent evaporation method11; the advantage in the melt method is that no organic solvents are needed.
Solid lipid particles have been proposed as a colloidal drug carrier therapeutic system for different administration routes such as oral, topical,12,13 ophthalmic, subcutaneous and intramuscular injection,9 and particularly for parenteral administration.14,15
Several sustained-release systems that include liposome16-18 and other biodegradable microspheres19,20 have been investigated as potential pulmonary carriers.
Until now the solid lipid microparticle (SLM) system has not yet been fully exploited for pulmonary drug delivery; little has been published in this area. The solid lipid particles might be used for pulmonary delivery in aqueous dispersions by nebulization or in dry powder inhalers.7
The lungs can provide a very effective means of delivery for many drugs. Medical conditions such as asthma, chronic obstructive pulmonary disease (COPD), or cystic fibrosis have traditionally been treated by inhaled drug delivery to the airways.
After administration to the airways, lipids may be eliminated by the common mechanisms for removal of carriers from the lungs, which are mucociliary transport, phagocytosis, or systemic absorption.21
The particle size and the potential toxicity of excipients are 2 of the critical factors that limit SLM use for pulmonary administration.7,22
The broadest and deepest penetration of particles into the airways and their deposition in the peripheral regions are achieved when the particle size ranges between 1 and 5 ?m.23
The excipients must be physiologically acceptable, biodegradable, and nonimmugenic and might not induce inflammatory and alloreactive responses.24,25
Solid lipid particles are well tolerated in living systems because they are made from physiological or physiologically related materials, therefore metabolic pathways exist; this finding is supported by in vitro studies of cytotoxicity and biodegradation.26,27 However, only a few in vivo studies have been performed as yet; and, in particular, toxicity studies after pulmonary administration should be investigated.
Among the screening methods allowing assessment of the presence of an inflammatory process occurring within the lower airways figures the cytological analysis of bronchoalveolar lavage fluid (BALF). By careful instillation of fluid into the airways, epithelial lining fluid and cellular components might be successfully recovered and analyzed. An increase of the total cell number in lavage fluid and modifications of the cellular population (eg, increase in polymorphonuclear neutrophils, in macrophages, etc) are considered as unspecific, but sensitive markers of an inflammatory reaction occurring within the lavaged lung parts.
Sterilization of microparticles should be taken into account in the case of pulmonary or parenteral administration. As reported in literature, the lipid particles are physically stable during sterilization by autoclaving.28,29,4
The aims of this study were the following:
investigation of the optimal conditions to produce SLM with a suitable diameter for penetration into the lower airways and to study the parameters affecting their preparation process.
characterization in terms of morphology (shape and surface) and size of SLM produced.
determination of SLM stability after lyophilization and sterilization by autoclaving processes.
assessment of the acute pulmonary toxicity of SLM in vivo by analyzing BALF after intratracheal instillation of SLM dispersions in rats.
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