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Abstract
Here we present a simple technique for re-directing reactions on the cell surface to the outermost region of the glycocalyx. Macromolecular crowding with inert polymers was utilized to reversibly alter the accessibility of glycocalyx proteoglycans toward cell-surface reactive probes allowing for reactivity control in the longitudinal direction (‘z’-direction) on the glycocalyx. Studies in HUVECs demonstrated an oncotically driven collapse of the glycocalyx brush structure in the presence of crowders as the mechanism responsible for re-directing reactivity. This phenomenon is consistent across a variety of macromolecular agents including polymers, protein markers and antibodies which all displayed enhanced binding to the outermost surface of multiple cell types. We then demonstrated the biological significance of the technique by increasing the camouflage of red blood cell surface antigens via a crowding-enhanced attachment of voluminous polymers to the exterior of the glycocalyx. The accessibility to Rhesus D (RhD) and CD47 proteins on the cell surface was significantly decreased in crowding-assisted polymer grafting in comparison to non-crowded conditions. This strategy is expected to generate new tools for controlled glycocalyx engineering, probing the glycocalyx structure and function, and improving the development of cell based therapies.
Highlights
When attaching macromolecules to the cell surface, both applications require a large stoichiometric excess of functionalized substrate to impart a cellular response[17,18]
When these substrates are used for cell surface engineering applications the components of the extracellular matrix are often preferentially modified as they are the first point of contact for protein-reactive substrates in the bulk solution
While hyperbranched polyglycerol (HPG) was the macromolecular agent used in the current study, in theory other more readily available hydrophilic polymers previously demonstrated as useful in macromolecular crowding may be used to the same effect[31]
Summary
When attaching macromolecules to the cell surface, both applications require a large stoichiometric excess of functionalized substrate to impart a cellular response[17,18]. We show that the use of inert macromolecular crowders in cell media can reversibly collapse the glycocalyx and enhance the binding of polymers, protein markers and antibodies to the structures’ outermost surface of multiple cell types. This methodology can be used to discriminately probe glycocalyx function in the ‘z’ direction or amplify the biological response of surface engineered cells, as we demonstrated in the improved immunocamouflage of engineered red blood cells
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