Investigating polyanions' binding to TSLP by SPR For an initial assessment of the influence of charge, size, flexibility and anionic group, the synthesized polyanions were first analyzed regarding their binding to TSLP. These results were compared to those observed with heparin and heparan sulfate (HS) using SPR. The synthetic polyglycerol sulfates showed remarkably strong binding to TSLP, outperforming the natural polymers heparin and heparan sulfate (Figure ). The calculated binding constants are shown in Table . Here, linear polyglycerol sulfate (lPGS10) showed the strongest binding with an equilibrium dissociation constant of KD = 0.565 ng/mL, while the dendritic variant with the same size of 9.9 kDa and equivalent degree of sulfation (90%) showed a 5-fold less affine binding with an equilibrium dissociation constant of KD = 2.9 ng/mL. Thus, the linear PG architecture is preferred, with the flexible presentation of sulfate groups allowing for more efficient chargecharge interactions. The polyglycerol carboxylates, by contrast, did not bind to TSLP. This effect has been observed previously with other proteins, suggesting that sulfates play a unique role in the binding process. When investigating the binding affinity of differently functionalized polyglycerols to L-selectin, we found that the interaction strength increased in the following order: carboxylate < phosphate < phosphonate = sulfonate < bisphosphonate < sulfate. This difference might result from variations in the polyanion protonation degree, since polycarboxylates have higher pKa values than polysulfates. This hypothesized effect did not lead to reduced zeta potentials of the carboxylates compared to the sulfates. This can be attributed to the fact that the zeta potential only describes the potential difference at the slipping plane rather than the direct surface of the polymer. We also observed a strong interaction of heparin with TSLP (KD = 16.5 ng/mL), while HS showed a weaker interaction ((KD = 3480 ng/mL). The weaker interaction of heparan sulfate with TSLP can be explained by its reduced number of negatively charged groups compared to heparin. In addition to their difference in molecular weight, heparin and heparan sulfate differ strongly in number of sulfate groups. While heparin bears approximately 1.4 to 2.5 sulfate groups per disaccharide unit, heparan sulfate contains just 0.55 to 1.25 sulfate groups per disaccharide unit. The weaker binding of heparin compared to the polyglycerol sulfates most likely results from the reduced density of sulfate groups, since carboxylate groups seem to be irrelevant for binding. While the lPGS10 presents 60 sulfate groups, heparin displays 54 sulfate groups (considering an average of 1.95 sulfate groups per disaccharide) over an increased molecular weight of 15 kDa, leading to a notable reduction of sulfate density. An increased number of functional groups also increases the number of counterions released during the binding process, and the resulting increase in entropy is the driving force of electrostatic interaction. The previously described role of hydrophobic interactions involved in protein binding of heparin may also contribute to the strong binding action we observed. PG itself did not show any binding, confirming that the binding interaction was derived from the polyanions' sulfate groups rather than their PG backbones.