Direct Cytosolic Delivery of Proteins Using Lyophilized and Reconstituted Polymer-Protein Assemblies

Purpose Cytosolic delivery of proteins accesses intracellular targets for chemotherapy and immunomodulation. Current delivery systems utilize inefficient endosomal pathways of uptake and escape that lead to degradation of delivered cargo. Cationic poly(oxanorbornene)imide (PONI) polymers enable highly efficient cytosolic delivery of co-engineered proteins, but aggregation and denaturation in solution limits shelf life. In the present study we evaluate polymer-protein nanocomposite vehicles as candidates for lyophilization and point-of-care resuspension to provide a transferrable technology for cytosolic protein delivery. Methods Self-assembled nanocomposites of engineered poly(glutamate)-tagged (E-tagged) proteins and guanidinium-functionalized PONI homopolymers were generated, lyophilized, and stored for 2 weeks. After reconstitution and delivery, cytosolic access of E-tagged GFP cargo (GFP E15 ) was assessed through diffuse cytosolic and nuclear fluorescence , and cell killing with chemotherapeutic enzyme Granzyme A (GrA E10 ). Efficiency was quantified between freshly prepared and lyophilized samples. Results Reconstituted nanocomposites retained key structural features of freshly prepared assemblies, with minimal loss of material. Cytosolic delivery (> 80% efficiency of freshly prepared nanocomposites) of GFP E15 was validated in several cell lines, with intracellular access validated and quantified through diffusion into the nucleus. Delivery of GrA E10 elicited significant tumorigenic cell death. Intracellular access of cytotoxic protein was validated through cell viability. Conclusion Reconstituted nanocomposites achieved efficient cytosolic delivery of protein cargo and demonstrated therapeutic applicability with delivery of GrA E10 . Overall, this strategy represents a versatile and highly translatable method for cytosolic delivery of proteins. Graphical Abstract