Design principles for cytokine-neutralizing gels: Cross-linking effects

Constructs composed of cytokine-neutralizing antibodies conjugated to high-molecular-weight hyaluronic acid have been shown to be effective at controlling inflammatory responses in vivo. A critical question in the development of this new class of biomaterial is whether crosslinked conjugates have similar anti-inflammatory effects, which would open up a broad range of tissue engineering applications in which the material would have intrinsic inflammation-controlling function. To test this, high-molecular-weight hyaluronic acid was conjugated with monoclonal antibodies to the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α in two forms of the material: viscous, non-crosslinked polymer-antibody conjugates and crosslinked, elastomeric polymer-antibody conjugates. The cytokine affinities of both constructs were validated using molecular characterization methods, and the biological activities were tested through subcutaneous implantation in Sprague-Dawley rats. In vitro, both forms of these constructs are capable of binding cytokines, but in vivo only the non-crosslinked polymer significantly reduces markers of acute inflammation compared to controls that lack the antibodies. We propose that these materials function by retarding cytokine diffusion, with the non-crosslinked polymers being capable of retarding the diffusion of cytokines in the extracellular matrix and preventing engagement with receptors. In contrast, crosslinked materials have long diffusion lengths into the gel compared with those between cells on the surface of the material, which may make them ineffective at sequestering pro-inflammatory cytokines on biologically relevant timescales. These results suggest an important design principle for preparing cytokine-regulating materials based on consideration of transport phenomena.