In Vitro Release Testing of PLGA Microspheres with Franz Diffusion Cells

In the present study, two methods were used to evaluate the in vitro release of leuprolide acetate (LA) from poly(lactide-co-glycolide) (PLGA) microspheres: Franz diffusion cells, typically referred to as “vertical diffusion cells” (VDC), and rotating bottle apparatus (RBA), both modified with a dialysis membrane. This hydrosoluble peptide was chosen as a model drug to study different possibilities of in vitro testing and analyze the variables that affect drug release, respecting sink and physiological conditions. Microspheres were prepared with a conventional double emulsion–solvent evaporation method using PLGA (50:50) with a relatively low molecular weight. Comprehensive stability tests for LA were performed in the conditions used for in vitro release assays. In phosphate-buffered saline (PBS), LA showed no significant degradation, but in an acidic medium, it degraded dramatically. The release profile of the delivery system was governed mainly by diffusion as explained by the low molecular weight of the polymer and the high water solubility of the peptide. The in vitro release profiles were triphasic in vertical diffusion cells and biphasic in the rotating bottle apparatus. The release kinetics was enhanced in RBA with respect to VDC, probably because the constant movement of a suspension of loose microspheres in a large volume and the large membrane area facilitated drug migration. The smoother, triphasic profiles obtained with VDC can be explained by the partial confinement of microspheres, which is similar to the described in vivo behavior of an injectable delivery system. INTRODUCTION The advent of modified-release delivery systems brought the complex issue of in vitro release evaluation, which has not yet been fully solved. Many devices and methods have been tested to clarify the matter and set specifications. Because not all dosage forms should fulfill the same requirements, this becomes even more difficult. For microspheres, much has been done, but there is still disagreement about the best in vitro release testing method to apply. Drugs and polymers of different natures, microsphere features, in vitro release devices, receptor media, and sink conditions are some of the issues that may be encountered during decisionmaking. Many authors have analyzed and discussed the suitability of different devices to perform the release tests of prolonged-release systems (1, 2). For instance, USP Apparatus 4, which is based on flow-through cells, was successfully used to obtain release profiles of dexamethasone from long-term, modified-release formulations (3). This is in accordance with USP recommendations about the suitability of this equipment for delivery systems containing drugs with limited solubility (4). Dialysis tests performed with different devices may be useful for testing biodegradable microspheres (5). Different types of shaking and rotating devices have also been widely used for this purpose (6). Franz diffusion cells, typically referred to as “vertical diffusion cells” (VDC), were initially intended for skin permeation. They were further modified to evaluate nasal inserts and other mucosal dispersed systems (7–9). Another attempt to develop in vitro tests for microspheres was the elevated temperature accelerated assay, which is useful mainly for batch-to-batch comparisons of longacting dosage forms, but they reflect neither the real-time release rate nor the involved mechanism (3, 10, 11). After the patent for the leuprolide-polylactic-co-glycolic acid (LA–PLGA) delivery system was issued in the late 1980s, it was studied extensively (12, 13). The release profile of the drug may be influenced by many parameters such as physicochemical properties and drug loading, variations of polymer molecular weight, lactic-to-glycolic ratio, microencapsulation conditions, and in vitro test protocols (14, 15). This particular system usually shows a triphasic release profile characterized by an initial burst of the drug near the surface or associated with pores after polymer wetting, usually defined as the amount released during the first 24 h (15), a lag phase until sufficient polymer erosion has taken place, and a secondary burst with approximately zero-order release kinetics (16–18). This feature generally applies for all cases, but polymer molecular weight, glass-transition temperature (Tg), drug properties, and even device geometry play important roles in precisely defining the release mechanism of a given delivery system. When a low molecular weight PLGA polymer is employed, for instance, the release is ruled mostly by diffusion. Zolnik and Burgess (19) explained that PLGA degrades from inside to outside at physiological pH. Degradation begins with water going inward; hydrolysis leads to the production of acidic oligomers, which are retained within the microspheres because of the relative hydrophobicity of the polymer, and the phenomenon finally influences the degradation mechanism. When PLGA *Corresponding author. diss-19-02-02.indd 6 5/21/2012 9:49:51 AM dx.doi.org/10.14227/DT190212P6