title: Noncovalent PEGylation by polyanion complexation as a means to stabilize keratinocyte growth factor-2 (KGF-2).
abstract: Repifermin, a truncated form of fibroblast growth factor-10 (FGF-10) also known as keratinocyte growth factor-2 (KGF-2), is a heparin-binding protein with potent regenerative properties. The protein unfolds and aggregates at relatively low temperature (~37 °C). Electrostatic interactions between polyanions and several FGFs have been reported to enhance the thermal stability of these proteins. Polyethylene glycol (PEG) was grafted to the polyanions pentosan polysulfate (PPS) and dextran sulfate (DS) as an alternative means to stabilize and noncovalently PEGylate KGF-2. Physical characteristics of KGF-2:polyanion-PEG complexes were examined using a variety of methods including circular dichroism (CD), intrinsic tryptophan fluorescence, differential scanning calorimetry, and dynamic light scattering. When compared to KGF-2 alone, subtle changes in CD spectra and fluorescence emission maxima were found when KGF-2 was formulated with the synthetic PEG-polyanions. Highly PEGylated polyanions (DS-PEG5) did not bind KGF-2 as well as conjugates with fewer PEG chains. The molecular weight of PEG did not have a noticeable effect on KGF-2 binding to the various PEG-polyanion conjugates. At optimal molar ratios, PPS-PEG and DS-PEG conjugates were able to stabilize KGF-2 by increasing the melting temperature by approximately 9-17 °C. Thus, polyanion-PEG conjugates improved the stability of KGF-2 and also offered a new electrostatic PEGylation scheme that may be extrapolated to other heparin-binding proteins.
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States.
authors.forename: Supang
authors.initials: S
authors.lastname: Khondee
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Olsen
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Zeng
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.forename: Cory
authors.initials: C
authors.lastname: Berkland
mesh_terms: D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003603:Cytotoxins / Q000494:pharmacology; D016264:Dextran Sulfate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology; D051526:Fibroblast Growth Factor 10 / Q000737:chemistry*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects; D006801:Humans; D008027:Light; D008970:Molecular Weight; D010316:Particle Size; D010426:Pentosan Sulfuric Polyester / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry*; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D012542:Scattering, Radiation; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: D003603:Cytotoxins; D051526:Fibroblast Growth Factor 10; D010426:Pentosan Sulfuric Polyester; D011092:Polyethylene Glycols; D016264:Dextran Sulfate