Polymer conjugated biologics form a multibillion dollar market, dominated by poly(ethylene glycol) (PEG). Recent reports linked PEGs to immunological concerns, fueling the need for alternative polymers. Therefore, we are presenting a strategy replacing PEG by poly(2-oxazoline) (POx) polymers using genetically engineered interleukin-4 (IL-4) featuring an unnatural amino acid for site-specific conjugation through bioorthogonal copper-catalyzed azide alkyne cycloaddition (CuAAC). Conjugation yields of IL-4-PEG were poor and did not respond to an increase in the copper catalyst. In contrast, POxylated IL-4 conjugates resulted in homogeneous conjugate outcome, as demonstrated electrophoretically by size exclusion chromatography and analytical ultracentrifugation. Furthermore, POxylation did not impair thermal and chemical stability, and preserved wild-type IL-4 activity for the conjugates as demonstrated by TF-1 cell proliferation and STAT-6 phosphorylation in HEK293T cells, respectively. In conclusion, POxylation provides an interesting alternative to PEGylation with superior outcome for the synthesis yield by CuAAC and resulting in conjugates with excellent thermal and chemical stress profiles as well as biological performances.
Driving macrophage (Mϕ) polarization into the M2 phenotype provides potential against inflammatory diseases. Interleukin-4 (IL-4) promotes polarization into the M2-Mϕ phenotype, but its systemic use is constrained by dose-limiting toxicity. Consequently, we developed IL-4-decorated surfaces aiming at sustained and localized activity. IL-4 muteins were generated by genetic code expansion; Lys42 was replaced by unnatural amino acids (uAAs). Both muteins showed cell-stimulation ability and binding affinity to IL4Rα similar to those of wt-IL-4. Copper-catalyzed (CuAAC) and copper-free strain-promoted (SPAAC) 1,3-dipolar azide-alkyne cycloadditions were used to site-selectively anchor IL-4 to agarose surfaces. These surfaces had sustained IL-4 activity, as demonstrated by TF-1 cell proliferation and M2, but not M1, polarization of M-CSF-generated human Mϕ. The approach provides a blueprint for the engineering of cytokine-activated surfaces profiled for sustained and spatially controlled activity.
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