David C Luther1, Harini Nagaraj1, Ritabrita Goswami1, Yağız Anıl Çiçek1, Taewon Jeon1,2, Sanjana Gopalakrishnan1, Vincent M Rotello3. 1. Department of Chemistry, University of Massachusetts, 379A LGRT Tower A, 710 North Pleasant St., Massachusetts, 01003, Amherst, USA. 2. Molecular and Cellular Biology Graduate Program, University of Massachusetts, 710 North Pleasant St., Massachusetts, 01003, Amherst, USA. 3. Department of Chemistry, University of Massachusetts, 379A LGRT Tower A, 710 North Pleasant St., Massachusetts, 01003, Amherst, USA. rotello@umass.edu.
Abstract
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 (GFPE15) was assessed through diffuse cytosolic and nuclear fluorescence, and cell killing with chemotherapeutic enzyme Granzyme A (GrAE10). 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 GFPE15 was validated in several cell lines, with intracellular access validated and quantified through diffusion into the nucleus. Delivery of GrAE10 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 GrAE10. Overall, this strategy represents a versatile and highly translatable method for cytosolic delivery of proteins.
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 (GFPE15) was assessed through diffuse cytosolic and nuclear fluorescence, and cell killing with chemotherapeutic enzyme Granzyme A (GrAE10). 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 GFPE15 was validated in several cell lines, with intracellular access validated and quantified through diffusion into the nucleus. Delivery of GrAE10 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 GrAE10. Overall, this strategy represents a versatile and highly translatable method for cytosolic delivery of proteins.
Authors: Ying Jiang; Joseph Hardie; Yuanchang Liu; Moumita Ray; Xiang Luo; Riddha Das; Ryan F Landis; Michelle E Farkas; Vincent M Rotello Journal: J Control Release Date: 2018-06-05 Impact factor: 9.776
Authors: Mario Vazdar; Jan Heyda; Philip E Mason; Giulio Tesei; Christoph Allolio; Mikael Lund; Pavel Jungwirth Journal: Acc Chem Res Date: 2018-05-25 Impact factor: 22.384
Authors: Federica Scaletti; Joseph Hardie; Yi-Wei Lee; David C Luther; Moumita Ray; Vincent M Rotello Journal: Chem Soc Rev Date: 2018-05-21 Impact factor: 54.564
Authors: Anselm F L Schneider; Antoine L D Wallabregue; Luise Franz; Christian P R Hackenberger Journal: Bioconjug Chem Date: 2019-01-14 Impact factor: 4.774