Multilayered Films Fabricated from an Oligoarginine-Conjugated Protein Promote Efficient Surface-Mediated Protein Transduction

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Abstract
The conjugation of cationic protein transduction domains to proteins results in an increase in the extent to which proteins are internalized by cells. This investigation sought to determine whether the conjugation of a protein transduction domain to a functional protein could be used to facilitate the incorporation of the protein into multilayered polyelectrolyte films and, subsequently, whether these films could be used to promote surface-mediated protein transduction. We demonstrate that it is possible to fabricate multilayered assemblies 80 nm thick using sodium polystyrene sulfonate (SPS) and bovine pancreatic ribonuclease (RNase A) conjugated to the cationic protein transduction domain nonaarginine (R9) using an entirely aqueous layer-by-layer process. We demonstrate further that the conjugation of R9 to RNase A permits the assembly of multilayered films under conditions that do not allow for the incorporation of the unmodified protein. This result suggests that R9 functions as a cationic anchor and serves to increase the strength of electrostatic interactions with SPS and facilitate layer-by-layer assembly. We also demonstrate that RNase A−R9/SPS films dissolve rapidly in physiologically relevant media and that macroscopic objects coated with these materials can be used to mediate high levels of protein transduction in mammalian cells. These results suggest the basis of general methods that could contribute to the design of materials that permit spatial and temporal control over the delivery of therapeutic proteins to cells and tissues.