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.

Protein–polymer therapeutics: a macromolecular perspective

“…103 On the other hand, Berkland and coworkers grafted a linear PEG (5 and 20 kDa) onto pentosan polysulfate (PPS), a polyanionic polymer, to induce electrostatic binding towards positively charged keratinocyte growth factor-2 (KGF-2). 104 Consistent with results for covalent PEG conjugates, the supramolecular construction of PEGylated proteins revealed increased thermal stability and higher hydrodynamic radii. 104 Recent interest in exploring various synthetic groups to promote supramolecular interactions has expanded the arsenal of chemical tools, with a major advancement in hostguest and boronic acid based chemistries.…”

“…104 Consistent with results for covalent PEG conjugates, the supramolecular construction of PEGylated proteins revealed increased thermal stability and higher hydrodynamic radii. 104 Recent interest in exploring various synthetic groups to promote supramolecular interactions has expanded the arsenal of chemical tools, with a major advancement in hostguest and boronic acid based chemistries. [105][106][107] Scherman and coworkers utilized the specificity of macrocyclic cucurbit- [8]uril towards electron deficient viologen and electron rich naphthalene to form a BSA-cucurbit [8]uril-PEG ternary complex.…”

Wrap-and-Strip Technology of Protein–Polyelectrolyte Complex for Biomedical Application

“…This type of specific binding has been reported for the other protein-PEGylated polymers, such as biotin-PEGylated peptide with avidin [84], PEGylated polyanions, pentosan polysulfate, and dextran sulfate toward a heparin-binding protein of repifermin [85].…”

A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor

“…For example, non-conjugated PEG-polyanions (pentosan polysulfate and dextran sulfate) have been utilized to increase stability of a related protein keratinocyte growth factor-2 (KGF-2); 49 histidine-tagged FGF-8a has been non-covalently associated with nitrilotriacetic acid-nickel-modified poly(acrylamide). 50 These complexes are non-covalent.…”