Noncovalent PEGylation by Polyanion Complexation as a Means To Stabilize Keratinocyte Growth Factor-2 (KGF-2)

• Published in: Biomacromolecules 2011-11, Vol.12 (11), p.3880-3894
• Main Authors: Khondee, Supang, Olsen, Christopher M., Zeng, Yuhong, Middaugh, C. Russell, Berkland, Cory
• Format: Article
• Language: English
• Subjects: Applied sciences; Biological and medical sciences; Calorimetry; Calorimetry, Differential Scanning; Cells, Cultured; Chromatography, Gel; Cytotoxins - pharmacology; Dextran Sulfate - analogs & derivatives; Dextran Sulfate - chemistry; Dextran Sulfate - pharmacology; Exact sciences and technology; Fibroblast Growth Factor 10 - chemistry; General pharmacology; Human Umbilical Vein Endothelial Cells - drug effects; Humans; Light; Medical sciences; Molecular Weight; Natural polymers; Particle Size; Pentosan Sulfuric Polyester - analogs & derivatives; Pentosan Sulfuric Polyester - chemistry; Pentosan Sulfuric Polyester - pharmacology; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Physicochemistry of polymers; Polyethylene Glycols - chemistry; Protein Stability; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Unfolding; Scattering, Radiation; Starch and polysaccharides; Thermodynamics; Titrimetry
• ISSN: 1525-7797; 1526-4602
• DOI: 10.1021/bm2007967

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.