title: Stabilization of alpha-chymotrypsin at the CH2Cl2/water interface and upon water-in-oil-in-water encapsulation in PLGA microspheres.
abstract: Protein inactivation and aggregation are serious drawbacks in the encapsulation of proteins in bioerodible polymers by water-in-oil-in-water (w/o/w) encapsulation. The model protein alpha-chymotrypsin was employed to investigate whether its stabilization towards the major stress factors in the w/o/w encapsulation procedure would allow for the encapsulation and release of non-aggregated and active protein. Due to the formation of amorphous aggregates alpha-chymotrypsin is an excellent sensor to probe unfolding events. Furthermore, its enzymatic activity is highly sensitive towards the presence of organic solvents. alpha-Chymotrypsin in aqueous solution showed substantial aggregation and activity loss when it was homogenized with CH(2)Cl(2) due to adsorption to the interface. Its w/o/w encapsulation in poly(lactic-co-glycolic)acid (PLGA) microspheres caused formation of 35% non-covalent aggregates and reduced the specific activity by 14%. Screening for efficient excipients revealed that co-dissolving the protein with maltose and polyethylene glycol (PEG, M(w) 5000) in the first aqueous phase reduced interface-induced protein aggregation and inactivation. Employing these excipients during encapsulation led to a reduction in alpha-chymotrypsin inactivation (10%) and aggregation (12%). Optimizing the effect of PEG by also dissolving the excipient in the organic phase prior to encapsulation further decreased the amount of non-covalent aggregates to 7% and loss in activity to 5%. The data obtained demonstrate that the w/o emulsification step is the main stress-factor in the w/o/w encapsulation procedure but subsequent encapsulation steps also cause some protein aggregation.
authors.affiliation: University of Puerto Rico, Río Piedras Campus, Department of Chemistry, P.O. Box 23346, San Juan, PR 00931-3346, USA.
authors.forename: Caroline
authors.initials: C
authors.lastname: Pérez-Rodriguez
authors.forename: Nashbly
authors.initials: N
authors.lastname: Montano
authors.forename: Karilys
authors.initials: K
authors.lastname: Gonzalez
authors.forename: Kai
authors.initials: K
authors.lastname: Griebenow
mesh_terms: D000818:Animals; D002214:Capsules; D002918:Chymotrypsin / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D045703:Emulsifying Agents / Q000737:chemistry; D004655:Emulsions / Q000737:chemistry; D004795:Enzyme Stability; D005612:Freeze Drying; D006016:Glycolates / Q000737:chemistry; D066298:In Vitro Techniques; D019344:Lactic Acid; D008320:Maltose / Q000493:pharmacokinetics; D008737:Methyl Chloride / Q000737:chemistry* / Q000493:pharmacokinetics; D008863:Microspheres; D011092:Polyethylene Glycols / Q000493:pharmacokinetics; D011098:Polyglactin 910 / Q000737:chemistry; D011100:Polyglycolic Acid; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011489:Protein Denaturation / Q000187:drug effects; D012710:Serum Albumin, Bovine; D013535:Suspensions; D014867:Water
chemical_list: D002214:Capsules; D045703:Emulsifying Agents; D004655:Emulsions; D006016:Glycolates; D013535:Suspensions; D014867:Water; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D012710:Serum Albumin, Bovine; D019344:Lactic Acid; D011098:Polyglactin 910; D011092:Polyethylene Glycols; D008320:Maltose; D008737:Methyl Chloride; D002918:Chymotrypsin; C117608:alpha-chymotrypsin