The influence of protein solubilisation, conformation and size on the burst release from poly(lactide-co-glycolide) microspheres

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Abstract

Encapsulation of proteins in poly(lactide-co-glycolide) microspheres via emulsion is known to cause insoluble protein aggregates. Following protein emulsification and encapsulation in PLGA microspheres, we used circular dichroism to show that the recoverable soluble protein fraction also suffers subtle conformational changes. For a panel of proteins selected on the basis of molecular size and structural class, conformational stability measured by chemical denaturation was not indicative of stability during emulsion-encapsulation. Partial loss of structure was observed for α-helical proteins released from freeze-dried microspheres in aqueous buffer, with dramatic loss of structure for a β-sandwich protein. The addition of sucrose (a lyoprotectant) did not prevent the loss of protein conformation upon encapsulation. Therefore, the conformational changes seen for the released soluble protein fraction originates during emulsification rather than microsphere freeze-drying. Analysis of the burst release for all proteins in buffer containing denaturant or surfactant showed that the degree of protein solubilisation was the dominant factor in determining the initial rate and extent of release. Our data for protein release into increasing concentrations of denaturing buffer suggest that the emulsion-denatured protein fraction remains insoluble; this fraction may represent the protein loss encountered upon comparison of protein encapsulated versus protein released.

Introduction

Poly(lactide-co-glycolide) (PLGA) microspheres are often employed to encapsulate proteins with the intention to bring about controlled release [1]. Unfortunately, a ‘burst release’ is commonly observed wherein the majority of protein (> 40%) is released from microspheres within the first 12 h [2], [3], [4], [5]. Release of the remaining protein may be observed during microsphere degradation but often a fraction of the protein encapsulated remains unaccounted for. Understanding the mechanism behind protein release will be critical if PLGA microspheres are to be used in therapeutic applications or tissue culture scaffolds. It is widely believed that the burst release represents solubilisation and diffusion of protein loosely associated with the surface of internal pores [6], [7]. This is in general agreement with encapsulation of fluorescently labelled proteins and visualisation by confocal laser scanning microscopy (CLSM), showing a heterogeneous protein distribution throughout the matrix [3], [4], [8]. Following the burst release, the subsequent rate of protein release is much reduced but may extend over 2 months or more, depending on the molecular weight of the protein [9], as well as microsphere size and erosion [10]. These studies suggest that the secondary release phase originates from protein entrapped within the polymer matrix which undergoes bulk erosion after homogenous dispersion of water throughout the microsphere.

Definitive interpretation of the release mechanism is complicated by the wide range of microsphere sizes and morphologies reported and the variety of proteins encapsulated. The distribution of drug, burst release and microsphere morphology has also been reported to be dependent upon the method of microsphere drying employed [11]. Further, imaging of microspheres following immersion in water shows development of a ‘skin’ with reduced external porosity after 5 h, and with near-complete closure of external pores after 24 h [2]. The mechanism is likely to involve plasticisation of the polymer glass as a result of lowering of the glass transition temperature upon microsphere hydration. Although this would correlate with the duration of some burst release periods reported, the number of external (‘exit’) pores for each microsphere appears to play little role in determining the protein release profile [4]. Complete closure of the external pores is therefore required, as demonstrated by attenuation of the burst release for protein encapsulated in the internal matrix of double-walled microspheres [12].

It is less clear how engineering of the protein itself, such as PEGylation, influences the burst release profile. Previous work has demonstrated that high molecular weight proteins are released from microspheres more slowly than low molecular weight proteins [9]. Similarly, one report has suggested that unfolded lysozyme conformers diffuse, by “reptation”, more rapidly out of microspheres than folded conformers [13]. This may be of relevance since proteins are well characterised as undergoing denaturation upon encapsulation, especially for emulsion-extraction fabrication [14], [15], [16]. However, it remains to be established if a wide range of different protein conformers are commonly released from microspheres, particularly if partially folded conformers existed in the matrix. In addition to ‘molten globule’ structures, partial unfolding may occur for modular proteins (such as immunoglobulin-type proteins) whose individual domains differ in conformational stability [17]. Although protein refolding back to a native conformation is possible, the extent to which this occurs during solubilisation of denatured protein in microspheres has not been previously investigated.

Here we investigate the fate of soluble and insoluble protein fractions following emulsification and encapsulation, and how the burst release is influenced by protein size, conformation and solubilisation upon microsphere wetting. To this aim a panel of proteins were used: the fibronectin 10th type III domain (FIII 10), chymotrypsinogen, bovine serum albumin (BSA), catalase and thyroglobulin. These proteins were selected since they represent α-helical and β-sheet (FIII10 and chymotrypsinogen) structural classes and progress in the order given by increasing molecular size. We have analysed protein conformation using circular dichroism to assess any structural changes for protein released from microspheres and the extent of refolding of denatured protein entrapped in microspheres. These data are complemented by size exclusion chromatography analysis of the change to protein hydrodynamic diameter following emulsification. We also assess the potential use of a preferentially excluded solute, sucrose, as a protein stabilising agent during lyophilisation.

Section snippets

Materials

BSA (fraction V), bovine liver catalase, bovine thyroid thyroglobulin, bovine pancreas α-chymotrypsinogen, lysozyme, poly vinylalcohol (PVA) (MW 22,000, 88% hydrolysed) and Triton X-100 were purchased from Sigma Chemical Company. PLGA copolymer (50 : 50 dl-lactide : glycolide, inherent viscosity 0.88 dl/g) was purchased from Purac Biochem, Netherlands. Water was purified to > 16 MΩ-cm. Dichloromethane (DCM) (analytical grade) was obtained from Fisher.

Expression and purification of FIII 10

Polyhistidine-tagged FIII 10 was expressed in E.

Chemical denaturation and burst release of proteins in denaturing media

Equilibrium chemical denaturation curves were taken as an indication of conformational stability (Fig. 1). The proteins unfolded via intermediate states such that two-state unfolding models enabling a quantitative comparison of stability could not be applied. Nonetheless, interpretation of the data with respect to previous detailed unfolding studies allowed sufficient interpretation for the purposes of this study. For thyroglobulin, step-wise upward shifts in Trp fluorescence were seen at 1.2 M

Discussion

With respect to the protein release data, we show that protein solubilisation is dominant in determining the extent of the burst release upon microsphere wetting. The morphology of the microspheres fabricated here did not appear to constrain physically the flux of protein through the external pores (cf. [13]); which were around 1–5 μm in diameter and roughly 3 orders of magnitude larger than the hydrodynamic radius of a 20 kDa protein [19]. Similarly, the tortuous nature of the internal porous

Conclusions

Loss of secondary structure for protein released from PLGA microspheres may be far more common than previously realised. It does not follow that release of soluble protein implies an intact native structure. Proteins with β-sandwich domains appear to be particularly susceptible to conformational loss during emulsification. In the case of β-sandwich proteins, emulsion-encapsulation in the presence of sucrose may be of benefit but optimisation of lyophilisation requires further work. The protein

Acknowledgement

We are indebted to Richard Oxland, University of Strathclyde Centre for Biophotonics, for technical support with CLSM. We would also like to thank the referees for helpful comments. This research was supported by the Wellcome Trust, grant number 067390/Z/02/Z, to CVDW and by the BBSRC to NCP for support of the CD facility.

References (33)

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