Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions

Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end.

ΔD (10 -6 ) B 0.004 % sHA B Figure S2. Adsorption of BSA on the PLL-g-PEG coated silica surface. PLL-g-PEG and BSA were dissolved in 10 mM HEPES buffer, pH 7.4 (B 1 ) and PBS (B 2 ), respectively. Introducing BSA to the system leads to a buffer effect but no binding after rinsing with buffer.

Comparison of adsorption profiles of different modification protocols of sHA.
In order to evaluate the evolution of the rigidity of sHA films, ΔD 7 /Δf 7 is plotted against time ( Figure S1). D/f plots provide a relative measure for the changes in the dissipation per unit added mass. 1 End-thiolated sHA exhibits a fast adsorption and quick saturation, which could be due to strong interactions between thiol groups and the gold surface. The most pronounced difference was seen for side-alkylated sHA adsorption, which exhibits a more dynamic profile and this could be a direct indication for the formation of a softer film. The sHA provided by LifeCore exhibits some degree of polydispersity, and side attachment could induce even higher apparent polydispersity, and subsequently larger variations in the density profile of the immobilized sHA, 2 which could lead to an altered hydration profile.

Multi-Parametric Surface Plasmon Resonance (MP-SPR) study to monitor thicknesses of the grafted sHA molecules on gold surfaces.
In order to validate the thickness values obtained from QCM-D for differently functionalized sHA layers, two-wavelength MP-SPR was employed. In this study, the samples were injected after 5 minutes of stable baseline after introducing the buffer (PBS) onto the gold sensors. The samples had different bulk refractive index compared to the background PBS. The bulk shift was corrected for using the PureKinetics™ software. Figure S4. Angle-time sensorgrams of a) EG 3 OH/EG 6 N 3, b) eSH-sHA (0.04 mg/mL) c) OEG/sidealkylated sHA and d) OEG/end-alkylated sHA evaluated by PureKinetics analysis. (The EG 6 N 3, and sHA content were adjusted to 67 mole percent). Black and red curves represent measurements at 670 nm and 785 nm, respectively.
In SPR, the shifts in the optical resonance properties of the sensor are correlated with the adsorbed materials and layer thickness. Applying two wavelengths in MP-SPR enables estimation of both refractive index and the thickness values by using Fresnel equations 3 , which is implemented in the BioNavis LayerSolver software. The Figure S4 shows an example fitting analysis for OEG/sidealkylated sHA before and after introducing the sample.

Control experiment for the adsorption of aggrecan and Lyve-1+Ab on EG 3 OH adlayer.
We investigated if aggrecan and Lyve-1+Ab complex would interact unspecifically with the oligo(ethylene) glycol layer when it is used to adjust the density of sHA. However, both aggrecan and Lyve-1+Ab do not induce any frequency or significant dissipation change on the EG 3 OH layer indicating that the surface is inert against aggrecan and Lyve1+Ab binding.

Comparison of the induced dissipation changes on sHA layers with different grafting densities.
Aggrecan binding was also probed at different densities of OEG/side&end-alkylated sHA. The results indicate qualitatively that a slight decrease in dissipation change was observed when OEG/side&endalkylated sHA density is lowered. In addition, the highest dissipation change was observed when the eSH-sHA high-density coverage was used.

Evaluation of Lyve-1+Antibody binding to side and end-alkylated sHA.
The semi-quantitative evaluation of the frequency and dissipation changes upon Lyve-1+Antibody and antibody binding on the side and end-alkylated surfaces are shown in Figure S8. As seen from the graphs, there is an apparent difference especially Lyve-1+Antibody vs antibody binding on these surfaces.