Protein microspheres as suitable devices for piroxicam release

Abstract

Bovine serum albumin-piroxicam (BSA-piroxicam) and human serum albumin-piroxicam (HSA-piroxicam) microspheres were sonochemically prepared and characterized. The use of polyvinyl alcohol (PVA) lead to an improvement of formulation characteristics, including smaller size, lower polydispersity index (PDl), higher entrapment efficiency and higher stability. The release kinetics of these proteinaceous microspheres was determined in presence of protease, indicating an anomalous drug transport mechanism (diffusion and polymer degradation). In presence of higher protease concentration, BSA microspheres exhibit Case II transport, leading to zero order release (protein degradation). These proteinaceous devices did not show cytotoxicity against human skin fibroblasts in vitro, for range concentrations below to 300 mg L⁻¹, greatly supporting their potential application in the treatment of inflammatory diseases.

Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs in the treatment of inflammatory diseases, such as rheumatic disease, but are also being increasingly used for non-rheumatic conditions, including acute and chronic pain, biliary and ureteric colic, dysmenorrheal inflammation and fever [1]. The mechanism of action of NSAIDs is mediated by its capacity to inhibit cyclo-oxygenase (COX) activity. The COX enzyme can be divided into two isoforms – a constitutive isoform (COX-1), which is responsible for maintaining normal function in the gastrointestinal and renal tracts, for example, and an inducible isoform (COX-2), which is found in areas of inflammation and in the brain [2]. It has been suggested that the anti-inflammatory actions of NSAIDs are due to the inhibition of COX-2, whereas the unwanted side-effects are due to the inhibition of COX-1 [3], [4], [5]. The vast majority of NSAIDs currently available is not selective for COX-2 and can thus cause the adverse reactions so commonly seen during NSAIDs treatment [3], [4], [5]. Therefore, the entrapment in microspheres of the NSAIDs, such as piroxicam, which are not selectively targeting COX-2, could be useful to minimize their adverse reactions. Various types of macromolecular substances, such as synthetic and natural polymers have been used in drug delivery research, as they can effectively deliver the drug to target the action site and thus increasing the therapeutic benefit, while minimizing the side effects [6], [7], [8], [9], [10]. Recently, protein microspheres have been shown efficacy as biodegradable and biocompatible carrier, which can incorporate a variety of drugs in relatively non-specific fashion [11], [12], [13], [14], [15]. Albumin is a promising material and has been extensively investigated as drug delivery system, because of its biodegradability and low toxicity [16], [17]. In addition, albumin proteins has a functionality for transporting different macromolecules in the bloodstream to target organs making it a potential macromolecular carrier for the site-directed delivery of drugs [18]. Four of the most important characteristics of particles are their size (presence of low polydispersity, bellow 0.1), entrapment efficiency, zeta-potential (surface charge), and the release characteristics [19]. Previous studies demonstrate that 3 min of sonication promote the entrapment of different components, such as antibiotic (tetracycline) [20], anti-cancer drugs (taxol and gemcitabine) [11], [12] or even RNA molecules [21] in bovine serum albumin (BSA) microspheres with approximately 2 μm of diameter with a high polydispersity index. However, the profile as well as the mechanism for the release of bioactive components at the targeted environment is not clarified. The main aim of this research was to study the efficiency of the sonochemical method for the entrapment of piroxicam in protein microspheres, using bovine or human serum albumin (HSA) as starting material, and to study the possibility of using the proteinaceous devices as a drug-delivery agent, determining their release kinetics in the presence of protease. Moreover, further miniaturization of the microspheres, to improve their colloidal stability and the entrapment efficiency of drug, was also performed via different stabilizers: polyvinyl alcohol (PVA), pluronic acid F68 (pluronic F68) and tween 80. Herein, this approach was applied in order to obtain particles with small diameters and a monodisperse population (polydispersity index lower than 0.1) increasing their surface area promoting at the same time a faster drug released. The ability of proteins to form microspheres was evaluated by quantifying protein concentration with the Lowry assay on the supernatant after ultrasound treatment [22]. Particle size, as well as the polydispersity index (PDl), of microspheres, was evaluated by photon correlation spectroscopy (PCS). It is well known that the particle size can significantly affect the microspheres properties and is important for their interaction with the biological environment [23]. Particle size values are thus crucial in the development and optimization of preparation process. In addition the zeta-potential of microspheres was also evaluated by electrophoretic laser Doppler anemometry, to obtain an indication of surface potential. The scanning transmission electron microscopy (STEM) has been employed to characterize the overall structure and shape of the microspheres. The entrapment efficiency of piroxicam in microspheres was assessed and the release profile was evaluated in the presence of a protease. Finally, microspheres were screened for cytotoxicity using a metabolic assay on an human fibroblast cell line.