Research paperProtein encapsulation and release from poly(lactide-co-glycolide) microspheres: effect of the protein and polymer properties and of the co-encapsulation of surfactants
Introduction
A wide range of proteins such as vaccines, cytokines, enzymes, hormones and growth factors are now commercially available in large quantity due to the recent advent of DNA technology. However, there are several problems associated with therapeutic trials of protein drugs. Among them are the short in vivo half-lives and the side effects attributable to the multiple and high-dose injections required to achieve desirable therapy. An interesting approach to prolong their therapeutic levels is to deliver these proteins via biodegradable polymers. In this sense, interest has been especially focused on the use of poly(dl-lactide-co-glycolide) (PLGA) microspheres as protein delivery systems 1, 2.
In order to successfully develop protein-loaded PLGA microspheres, it is essential that the biological activity of the protein is retained, not only throughout encapsulation but also during its prolonged release. However, the complex physical and chemical instabilities inherent to protein molecules, are obstacles for achieving this goal. In this sense, many peptides and proteins show a pronounced tendency to the self-association and adsorption process 3, 4, 5, 6, 7by electrostatic and hydrophobic mechanisms, forming high molecular weight aggregates of non-covalent or covalent nature [8]. These aggregates must be avoided, because they are frequently associated with a loss of protein activity.
Among the microencapsulation techniques, the o/o solvent evaporation [9]and the w/o/w solvent extraction techniques 10, 11, 12, 13, 14, 15are two of the most convenient ways for the encapsulation of proteins within PLGA microspheres. In both cases there is a minimal diffusion of the protein to the external phase. However, some authors have reported phenomena of protein aggregation and loss of activity following encapsulation by a w/o/w technique 16, 17, 18. To overcome this problem, one solution could be to use stabilisers, such as poloxamers, which have been shown to stabilise the primary emulsion and to reduce protein–polymer interactions [19].
Another important limitation still associated to PLGA microspheres as protein delivery systems is that protein release kinetics is often unpredictable. They commonly exhibit an initial burst of release followed by a very slow and incomplete release [20]. In this sense, it is surprising that, despite the high number of protein release studies performed until now, the effect of the protein properties and its interaction with the PLGA matrix on the in vitro protein release have scarcely been considered [21]. Previous studies have shown, however, that basic drugs can interact with acidic degradation products, during the PLGA degradation [22]. This fact suggests that proteins charged positively could interact with the PLGA degradation products thus hindering its release. Furthermore, the protein entrapped within the microspheres may experience a low environmental pH [23]due to the high local concentration of trapped degradation products, thus forming water insoluble non-covalently bound protein aggregates [24].
These previous results show the necessity of further investigating the simultaneous effect of the protein and polymer characteristics in protein encapsulation, as well as the need of searching for stabilisers which would preserve the protein stability during its encapsulation and further release. Taking this into account, in the present work we studied the influence of the following variables: type of microencapsulation procedure (w/o/w solvent extraction vs. o/o solvent evaporation), type of protein (BSA or LZM), PLGA molecular weight (Mw) (10 kDa or 34 kDa), and the incorporation or not of the stabilisers (poloxamer 188 or poloxamer 331) on the protein encapsulation efficiency and release from PLGA microspheres.
Section snippets
Materials
Bovine serum albumin (BSA, fraction V), fluorescein isothiocyanate labelled BSA (FITC-BSA) and lysozyme (LZM) were purchased from Sigma Chemical (Madrid, Spain). Poly(dl-lactide-co-glycolide) 50:50 (PLGA) copolymers with inherent viscosity (i.v.) of 0.16 and 0.4 dl/g were purchased from Birmingham Polymer (Birmingham, AL, USA) and Boehringer Ingelheim (Germany), respectively. Cottonseed oil was supplied by Sigma. The solvents ethyl acetate (EA), isopropyl alcohol and petroleum ether were
Results and discussion
The aim of this study was to investigate the mechanism of encapsulation of two model proteins, using two different microencapsulation procedures, and to elucidate the role of the protein properties on its encapsulation and release from PLGA microspheres. For this purpose two model proteins, BSA and LZM, which differ in their isoelectric point (4.6 and 11.2, respectively) and molecular weight (66 200 and 14 000 Da, respectively) were selected. They were encapsulated in two kinds of PLGA of low
Conclusions
This study shows the effect of the polymer–protein interactions on the inner structure, encapsulation efficiency and protein release from PLGA microspheres. These interactions were found to be dependent not only on the specific properties of the protein and the polymer, but also on the microencapsulation process and the co-encapsulation of the tensoactives, i.e. poloxamers. In a general sense, the more important the interaction is, the higher the protein loading and the slower the protein
Acknowledgements
This work was supported by grants from the Spanish Commission of Science and Technology (C.I.C.Y.T) (FAR91-0664 and SAF94-0579).
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2021, ChemCitation Excerpt :Until now, studies of molecular confinement in synthetic hosts have mainly focused on small molecules,1,2 owing to the experimental difficulties in synthesizing larger hosts. Encapsulation of protein molecules has previously been achieved with polymeric matrices, including micelles,3 vesicles,4 and polymer capsules.5 However, the broad distribution in molecular weight or aggregation number of such polymeric matrices results in polymeric hosts with nonuniform size and form.