Structure Activity Relationship of Heparin Mimicking Polymer p(SS- co-PEGMA): Effect of Sulfonation and Polymer Size on FGF2-Receptor Binding

Abstract

Fibroblast growth factor-2 (FGF2) is a heparin binding protein that plays a role in a range of biological functions such as wound healing and bone regeneration. Heparin, a highly sulfated glycosaminoglycan, is required for FGF2 to bind to its receptor. Therefore, polymeric mimics of heparin are widely studied for their ability to manipulate FGF2-induced biological interactions. It is known that altering the degree of sulfonated monomer incorporation and size of heparin-mimicking polymers can affect protein-receptor binding. To elucidate the relationship between degree of sulfonation and receptor binding for the heparin-mimicking polymer, poly(styrene sulfonate-co-poly(ethylene glycol) methyl ether methacrylate) (p(SS-co-PEGMA)) a library was synthesized to contain nine polymers with degrees of sulfonation ranging from 0-100%. Kinetics of the polymerization was evaluated and reactivity ratios compared to literature results. These polymers were then 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. Therefore, another library of polymers was prepared maintaining the degree of sulfonation at 81% and changing the size from 41 to 390 monomer repeat units. The polymers were again tested in receptor based ELISA and cell studies, and all of the different sizes performed similarly, except for degree of polymerization 295 and 390, which had reduced response in the cellular assay. These results provide important information for the use of pSS-co-PEGMA as a potential heparin-mimicking therapeutic.

Figure 1

Polymer kinetics performed on p(SS-co-PEGMA) polymerizations. (a) Kinetic studies of the RAFT polymerization of p(SS-co-PEGMA)₈₁ at 70 °C. Conversion, M₀, and M_t were calculated using ¹H NMR spectroscopy. (b) Percent styrene sulfonate incorporation over time. (c) Theoretical versus experimental M_n from right angle light scattering in aqueous running buffer. (d) F_SS versus f_SS curves.

Figure 2

SDS-PAGE with Western Blot of FGF-p(SS-co-PEGMA) conjugates in both (a) non-reducing conditions and (b) reducing conditions. Lane 1: FGF2, Lane 2: FGF2-p(SS-co-PEGMA)_8.0, Lane 3: FGF2-p(SS-co-PEGMA)₂₈, Lane 5: FGF2-p(SS-co-PEGMA)₅₅, Lane 6: FGF2-p(SS-co-PEGMA)₈₁, Lane 7: FGF2-pPEGMA, Lane 8: FGF2-p(SS-co-PEGMA)₇₆, Lane 9: FGF2-p(SS-co-PEGMA)₉₅, Lane 10: FGF2-pSS.

Figure 3

FGF2-p(SS-co-PEGMA) conjugates in FGF2 receptor binding assays. (a) FGF Receptor based ELISA. The experiment was repeated three times with n = 3 for each experiment. Error bars represent the standard deviation. (b) Cell growth of heparin-mimicking polymer conjugates in BaF3-FR1C cells. Data was normalized to the blank medium group, which was set at 100%. Each sample contained six replicates and the experiment was repeated three times. Error bars represent standard deviation. Statistical analysis was done using Student's t test. * p< 0.01 compared to FGF2, # p < 0.03 compared to FGF2.

Figure 4

Determination of FGF2-p(SS-co-PEGMA)₈₁ receptor binding in BaF3-FR1c with the addition of 1 μg/mL heparin. Data was normalized to the blank medium group, which was set at 100%. Each sample contained six replicates. Error bars represent standard deviation. Statistical analysis was done using Student's t test. * p< 0.01 compared to FGF2.

Figure 5

Examination of the ability of p(SS-co-PEGMA) to enhance FGF2 receptor binding. (a) FGF Receptor based ELISA. The experiment was repeated 3 times with n = 3 for each experiment. Error bars represent the standard deviation. (b) Cell growth of heparin-mimicking polymers in BaF3-FR1C cells. Data was normalized to the blank medium group, which was set at 100%. The experiment was repeated 3 times with n = 6 for each experiment. Error bars represent the standard deviation. Statistical analysis was done using Student's t test. * p< 0.01 compared to FGF2.

Figure 6

Light scattering GPC traces showing varying molecular weight polymers with 81% SS

Figure 7

Determining the ability of p(SS-co-PEGMA) with varying polymer sizes to enhance FGF receptor binding. (a) FGF receptor based ELISA. The experiment was repeated 3 times with n = 3 for each experiment. Error bars represent standard deviation. (b) Cell growth of heparin-mimicking polymers in BaF3-FR1C cells. Data was normalized to the blank medium group, which was set at 100%. Each sample contained 6 replicates and the experiment was repeated 2 times. Error bars represent standard error of the mean (SEM). Statistical analysis was done using Student's t test. * p< 0.01 compared to FGF2.

Scheme 1

Synthesis of heparin mimicking polymer-protein conjugate, FGF2-p(SS-co-PEGMA). (a) Polymerization of p(SS-co-PEGMA) via RAFT polymerization. (b) Conjugation of p(SS-co-PEGMA) to FGF2 via disulfide exchange. Protein structure modified from PDB 1CVS using PyMOL software.