Acidic Fibroblast Growth Factor Characterization

Introduction to Acidic Fibroblast Growth Factor

The Acidic Fibroblast Growth Factor (aFGF or FGF-1) is a protein, weighing approximately 16-kDa, that is found in numerous tissues. This protein exhibits a wide array of biological activities and is part of a group of at least nine known proteins with similar structure, collectively termed as fibroblast growth factors (FGFs). One of the distinguishing features of these proteins is their affinity towards sulfated polysaccharides like heparin and heparan proteoglycans, which are present on the majority of cell surfaces and essentially all basement membranes.

Fig 1. Endogenous levels of aFGF in T2DM mice and SCs. (Li, Rui, et al. 2021)

The FGF family includes two well-studied members, namely acidic (aFGF or FGF-1) and basic (bFGF or FGF-2) fibroblast growth factors. Both of these proteins serve as powerful mitogens for mesoderm-derived cells as well as several cells that originate from the embryonic ectoderm. Furthermore, aFGF and bFGF exert a chemotactic effect on cells in culture.

Turbidimetric Monitoring of aFGF Stability

When an aFGF solution is subjected to temperatures close to or greater than its transition midpoint (Tm) - the temperature at which the protein goes from a folded to unfolded state - the growth factor undergoes denaturation, losing its native tertiary structure. This denatured form of aFGF exhibits extreme insolubility and undergoes rapid aggregation as the unfolded protein emerges. Therefore, a convenient method to assess the structural stability of aFGF is to monitor the kinetics of temperature-induced aggregation by measuring the turbidity (degree of light scattering) at a wavelength of 350 nm.

Fig 2. Turbidity measurements of the heat-induced aggregation of aFGF in the presence of heparin. (Tsai, P. K., et al. 1993)

Delivery of aFGF as a Topical Agent

Developing aFGF into a therapeutic agent for wound healing involves more intricate considerations than merely ensuring its physicochemical and biological stability during storage. A key requisite involves the easy applicability of this biologically active growth factor to the wound in a clinical context, along with successful delivery and maintenance at the targeted tissue area.

To prevent run- off, a viscous preparation was considered appropriate provided it (1) does not interfere with bioactivity of the protein, (2) can withstand freezing at –70°C without loss of viscous properties, (3) is compatible with buffer components, (4) retains bioactivity of the protein after drying on the treatment area, (5) does not interfere with bioanalytical capabilities, and (6) can withstand sterilization.

Effect of Other Polyanions on aFGF Stability

To investigate the ability of polyanions other than heparin to stabilize acidic fibroblast growth factor (aFGF), a variety of composites analogous to heparin, including sulfated polysaccharides, sulfated and phosphorylated small molecules, and other strongly charged compounds, were investigated using turbidimetric methods at 40°C. The results of these investigations are summarized below. The ability of heparin analogs, such as sulfated polymers, to stabilize aFGF varies widely.

Fig 3. Stabilization by polyanions of aFGF against heat-induced aggregation at 40°C. (Tsai, P. K., et al. 1993)

For example, heparin, pentosan sulfate, and dextran sulfate showed greater efficacy (no aggregates manifested after 30 minutes) compared with chondroitin sulfate (which showed 10- to 100-fold stabilization), which in turn showed greater efficacy (ranging from unstable to 1- to 10-fold stabilized) than either polyethylene sulfate or carrageenan sulfate.

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References

1. Li, Rui, et al. Acidic fibroblast growth factor attenuates type 2 diabetes-induced demyelination via suppressing oxidative stress damage. Cell death & disease. 2021, 12.1: 107.
2. Tsai, P. K., et al. Formulation design of acidic fibroblast growth factor. Pharmaceutical research. 1993, 10: 649-659.

For research use only, not intended for any clinical use.