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Open Access Publications from the University of California

Heparin Mimicking Polymer Fibroblast Growth Factor 2 Conjugates for Improved Therapeutics

  • Author(s): Paluck, Samantha Joy
  • Advisor(s): Maynard, Heather D
  • et al.

Heparin is a naturally occurring highly sulfated polysaccharide that plays a critical role in a range of different biological processes. Due to the diverse functions of this polysaccharide in the body and drawbacks of its use, analogous heparin-mimicking materials are widely studied for therapeutic applications. Fibroblast growth factor 2 (FGF2) is a heparin-binding protein involved in cellular functions in applications such as wound healing and tissue regeneration. Stabilization of this protein is important for its use as a therapeutic since the native protein is unstable during storage and delivery. Additionally, the ability to increase the activity of FGF2 is important for its application, particularly in chronic wound healing and the treatment of various ischemic conditions. This dissertation focuses on the development of a new superagonist heparin-mimicking block copolymer FGF2 conjugate, the structure activity relationship of one of the polymer blocks, and the use of that polymer to covalently dimerize FGF2.

In Chapter 1, the synthesis and biological activity of heparin-mimicking polymers are outlined. Utilization of these polymers provides significant benefits compared to heparin, including enhancing therapeutic efficacy and reducing side effects as a result of fine-tuning heparin-binding motifs and other molecular characteristics. The major types of synthetic heparin-mimicking polymers are summarized in this chapter, as well as their applications. In Chapter 2 we report a heparin mimicking block copolymer, p(SS-co-PEGMA)-b-VS, that contains one segment that enhances the stability of FGF2 and one that binds to the FGF2 receptor. The FGF2 conjugate retained activity after exposure to refrigeration (4 �C) and room temperature (23 �C) for 7 days, while unmodified FGF2 was inactive after these standard storage conditions. A cell study and receptor-based enzyme-linked immunosorbant assay (ELISA) indicated that the conjugated block copolymer facilitated binding of FGF2 to its receptor to the same extent as the addition of heparin to FGF2. Furthermore, the conjugate increased the migration and angiogenesis of endothelial cells when compared to FGF2 alone.

In Chapter 3 the structure activity relationship of the stabilizing block, p(SS-co-PEGMA), was examined. A library was synthesized to contain nine polymers with degrees of sulfonation ranging from 0-100%. These polymers were tested for their ability to enhance FGF2 binding with its receptor as both covalent conjugates and as excipients. In a receptor based enzyme-linked immunosorbant assay (ELISA), as well as a cell-based study, the polymer with 81% SS incorporation enhanced receptor binding compared to FGF2 alone, and to a greater extent than the other polymers. Furthermore, a polymer size study did not show any further increase in receptor binding in relation to polymer chain length. These results provide important information for the use of p(SS-co-PEGMA) as a potential heparin-mimicking therapeutic.

In its active form, FGF2 is dimerized, typically with heparan sulfate threaded through the heparin binding domain. In Chapter 4 we show that we are able to increase the in vitro angiogenesis activity of FGF2 by covalently dimerizing it with 2 kDa poly(ethylene glycol) (PEG). We hypothesized that replacing the PEG linker with a heparin-mimicking polymer could increase the biological activity of the dimer. To fabricate a FGF2 dimer with heparin-mimicking linker, p(SS-co-PEGMA) was functionalized to contain maleimide end groups. The polymer was used to successfully dimerize FGF2. The purification of the dimer from unconjugated protein proved difficult and in the future we hope to purify the conjugate and asses its ability to stabilize FGF2 and increase the biological activity of the protein.

While bio-mimicking polymers provide unique characteristics for conjugates, the most commonly used polymer for protein conjugation is PEG. The covalent attachment of PEG to proteins (PEGylation) improves protein stability and bioavailability. However, there are still some downfalls to using PEG, including the non-degradable nature of the protein. Fabricating PEG-like polymers with degradable backbones is one way to overcome problems with bioaccumulation. In Chapter 5 we report the step growth polymerization of PEG-like polymers containing degradable oxime and hydrazone linkages. The polymers were shown to be degradable over 12 months with hydrazone linkages fully degrading, and oxime linkages partially degrading.

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