Development and characterization of protein-loaded poly(lactide-co-glycolide) nanospheres

Abstract

The goal of this work is to develop poly(lactide-co-glycolide) (PLGA) nanospheres designed to deliver proteins for extended periods of time. To accomplish this goal a water-in-oil-in-water emulsion technique was conveniently modified. A study was performed to evaluate how the solvent elimination procedure, the copolymer type (different molecular weight and containing either free or esterified carboxyls) and the surfactant Poloxamer 188 affected the properties of the nanoparticles. The size of the nanospheres become larger (from 300 to 600 nm) by increasing the copolymer molecular weight and by the incorporation of Poloxamer 188. The protein loading efficiency varied from 40 to 90%, reaching the maximum values for the formulations made of PLGA with free carboxyls. However, the co-encapsulation of Poloxamer 188 reduced the protein loading. The in vitro protein release rate, was fairly constant after an initial burst release. The release rate was significantly reduced for the copolymer with terminal free carboxyls but was enhanced by the incorporation of Poloxamer 188 in the nanoparticles. No significant aggregation or fragmentation of the encapsulated protein was observed after incubation for 1 month. Consequently, these nanoparticles can be proposed as new controlled release protein delivery systems.

Introduction

At present, protein delivery is a very promising area of research, due to the recognised necessity of improving the in vivo efficacy of the newly developed therapeutic as well as antigenic proteins. Among the protein delivery systems intended for parenteral administration, the poly(lactide-co-glycolide) (PLGA) microspheres, have been shown to have an important potential because of their ability to control the release of model and antigenic proteins 1, 2. Furthermore, these microspheres have also been revealed as promising carriers for the transport of antigens through the nasal and oral epithelia 3, 4, 5. In this latter sense, several authors 6, 7have shown, a number of years ago, that the size of the microspheres is a crucial parameter since it determines the uptake of the encapsulated antigen by the immune system. More specifically, their studies evidenced that microspheres less than 10 μm were preferable to those of a larger size in terms of their improved uptake by the Peyer's patches and further antigen presentation to the immune system. More recently, Jani et al. 8, 9, 10and Jenkins et al. [11]published an important work dealing with the oral uptake and in vivo distribution of polystyrene particles. These authors showed that the uptake of the nanospheres is much greater than that of the microspheres. In addition, they observed that the in vivo distribution of the particles which were taken up by the Peyer's patches was highly affected by their size. These results indicate that nanospheres are expected to disseminate systemically whereas microspheres are expected to remain and deliver their content in the Peyer's patches. Despite this information, which shows the great potential of the nanospheres as protein delivery systems for mucosal administration, it is surprising that there is no relevant published work dealing with the encapsulation and controlled release of hydrophilic proteins from PLGA nanospheres. In this context, it is worthwhile to mention that the techniques described in the literature for the production of PLGA nanoparticles involve the dissolution of the active compound in an organic solvent, a method that is not applicable, in general, for the encapsulation of proteins [12].

On the other hand, looking at the factors that govern the release of proteins from PLA and PLGA microspheres, it has been generally accepted that the polymer degradation rate substantially affects the release rate of the entrapped protein 13, 14, 15, 16, 17. However, almost no attention has been paid to the protein–polymer affinity and its consequences on the in vitro protein release rate. Our group has, very recently, reported that the use of new PLGA copolymers with free carboxylic end groups may drastically modify the in vitro release of the entrapped protein [18]. These results show the necessity of further investigation of the mechanisms and factors which control the release of proteins from PLGA-based systems.

Taking into account this information, the primary goal of this work has been to create PLGA nanospheres with an important capacity for the association and controlled release of proteins. Another purpose of this work was to improve the knowledge of the complex mechanisms that govern the release of proteins from PLGA systems. With this objective in mind, we used the bovine serum albumin (BSA) as a model protein and various PLGA copolymers differing in their molecular weight and also in the esterification of the terminal carboxyl groups of the polymer chain.