Molecular Mechanism of Improved Structural Integrity of Protein in Polymer Based Microsphere Delivery System Sanjay Rawat, Nandita Kohli, C. Raman Suri, and Debendra K. Sahoo* CSIRInstitute of Microbial Technology, Sector 39-A, Chandigarh 160036, India * S Supporting Information ABSTRACT: Polymer-based delivery systems provide a promising alter- native to multidose intake of many drugs/vaccines. Protein aggregation and inactivation, however, are major problems associated with the encapsulation of proteins in microspheres. With this in mind, we investigated the structural integrity of a model protein bovine serum albumin (BSA) released from poly(lactide-co-glycolide) (PLGA) based microspheres. BSA was encapsu- lated using solid-in-oil-in-water (S/O/W) double emulsification method with different mixtures of surfactants (carboxymethyl cellulose (CMC):Tween 20/CMC:Tween 80/Tween 20:Tween 80) and with or without polyethylene glycol (PEG). The morphology of BSA-loaded microspheres was analyzed using dynamic light scattering (DLS) and scanning electron microscopy (SEM). BSA released from lyophilized microspheres was evaluated for the structural, conformational and thermal stability by using various spectro- scopic and calorimetric techniques. Conformational analysis showed greater increase in secondary structural content of BSA in sample containing PEG and surfactant mixture of CMC and Tween 20 as compared to that containing other two mixtures of surfactants. The differential scanning calorimetric (DSC) analysis of released BSA from all PEG containing samples showed an increase in thermal stability of the protein. Furthermore, fluorescence spectra showed compactness of BSA. In conclusion our studies suggest macromolecular crowding, molecular confinement and increase in Gibbs free energy with strong electrostatic forces of repulsion between microspheres, the last phenomenon due to chosen surfactants, to be responsible for making the protein more compact and structurally integrated and result in a potential encapsulation process for improved protein integrity in final formulation. KEYWORDS: polyethylene glycol, surfactant, compactness, macromolecular crowding, molecular confinement, protein integrity I. INTRODUCTION Polymer-based drug and vaccine delivery systems provide a viable alternative to multidose intake of many drugs/vaccines. Several microencapsulation methods including those based on a solid-in-oil-in-water (S/O/W) double emulsification method have been proposed in which protein drugs are often applied as solid particles such as the preparation of biodegradable poly(lactide-co-glycolide) (PLGA) based horseradish peroxides and bovine superoxide dismutase. 1 However, most protein drugs have various stability problems. Hence, while developing a protein delivery system one must minimize their denaturation and aggregation during processing. 2-4 The encapsulation of protein pharmaceuticals in micro/nanospheres comprising the biodegradable and biocompatible polyester PLGA has been widely investigated for their sustained delivery. 5 This S/O/W method may be superior to the water-in-oil-in-water (W/O/W) emulsion method because solid-state proteins retain their structural integrity in organic solvents due to the kinetic trapping and reduction in contact with the organic phase such as the case of PLGA blended with PEG. Apart from its osmolytic effect PEG acts as a micronizing and emulsifying agent during the emulsifiaction process 1,2 and improves protein stability and delivery capacity. 6-9 The presence of PEG in the microspheres is also known to modify both the structure and hydrophilicity of the polymeric matrix and consequently the release profile. 13 It has been shown that addition of PEG 400 into the aqueous phase of the primary emulsion was effective in stabilizing a poorly stable protein, nerve growth factor (NGF), due to decrease in adsorption of the protein at the interface. 10-12 Earlier, we had reported that no significant changes in BSA conformation occurred by processes like lyophilization and sonication, when PEG 8000 was used as a stabilizer at an optimum BSA:PEG molar ratio. This molar ratio of BSA:PEG was found to stabilize BSA due to the protection of both the surface and buried hydrophobic core residues. 14 The incorporation of PEG into the emulsification process for making nano/microspheres also involves the interfacial phenomenon which is very crowded in nature due to high concentration of different additives along with PEG (macro- molecular components), which makes the interfacial environ- ment crowded in nature. 15-17 It is well reported that Received: October 4, 2010 Revised: June 20, 2012 Accepted: June 22, 2012 Published: June 22, 2012 Article pubs.acs.org/molecularpharmaceutics © 2012 American Chemical Society 2403 dx.doi.org/10.1021/mp2004065 | Mol. Pharmaceutics 2012, 9, 2403-2414