Could Diphtheria toxoid be protected from damages caused by water/methylene chloride interface during microencapsulation process?
Jocimara A.M. Namur1,2, Célia S. Takata1 and Maria Helena Bueno da Costa1*
1Lab. de Microesferas e Lipossomas- Centro de Biotecnologia- Instituto Butantan,
Av. Vital Brazil 1500 – 05503-900 Butantan *bdacosta@edu.usp.br and
2I. Química -USP, Av. Lineu Prestes, São Paulo, SP, Brasil
Water-in-oil-in-water [(W1/O)/W2] emulsions are commonly used to encapsulate water-soluble proteins into a PLGA [poly (lactide-co-glycolide acid)] polymeric matrixes. The W1/O step, when the protein has a contact with CH2Cl2 phase, is considered to be the major cause of protein denaturation and aggregation. We investigate, here, the conformation of the diphtheria toxoid (Dtxd), during the primary emulsion phase of its microencapsulation into PLGA microspheres. We investigate (by ultraviolet (uv), circular dichroism (CD), fluorescence spectroscopies, HPLC, and ELISA) how the Hofmeister ion series could interfere on protein stability during this step of microencapsulation process.
HPLC analysis demonstrated that after Dtxd emulsification in the absence of salt 35% of protein was aggregated. The percentage of protein aggregation increased to 60-70% in the presence of the cosmotropics NaH2PO4 or NaCl, and to 80% in the presence of the caotropic MgCl2. Unexpectedly only 5% of the Dtxd aggregated in the presence of the caotropic KSCN. The α-helical content of the protein in these preparations, as estimated by circular dicroism, were quite stable suggesting that Dtxd encapsulation is followed by protein aggregation with minor exposition of hydrophobic residues. The hydrophobic residues exposition, estimated by fluorescence, was pH-dependent and was higher in the presence MgCl2. In the presence of 10mM KSCN the Dtxd remained 95% soluble and with the same native conformation. The technological implications of these results are that the use of this simple salt may be sufficient to protect protein during PLGA microencapsulation and thus eliminate the requirement of other proteins, polyols and surfactants normally used stabilizers during this process. Grants: FAPESP (00/10970-7), CNPq (474781/01-1) and Fundação Butantan. JAM Namur had a fellowship from CNPq.
|