4.8 Article

Guanidyl-Rich Poly(ß Amino Ester)s for Universal Functional Cytosolic Protein Delivery and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 Ribonucleoprotein Based Gene Editing

Journal

ACS NANO
Volume 17, Issue 18, Pages 17799-17810

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acsnano.3c03269

Keywords

cytosolic protein delivery; poly(ss amino ester)s; guanidyl; CRISPR/Cas9 ribonucleoprotein transfection; gene editing

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Protein therapeutics show great promise for treating complex diseases, but the lack of suitable delivery vectors limits their clinical use, particularly for delivering functional cytosolic proteins in vivo. This study demonstrates that modifying poly(ss amino ester)s (PAEs) with a phenyl guanidine (PG) group enhances their capabilities for intracellular delivery of cytosolic functional proteins and CRISPR/Cas9 ribonucleoprotein.
Protein therapeutics are highly promising for complex disease treatment. However, the lack of ideal delivery vectors impedes their clinical use, especially the carriers for in vivo delivery of functional cytosolic protein. In this study, we modified poly(ss amino ester)s (PAEs) with a phenyl guanidine (PG) group to enhance their suitability for cytosolic protein delivery. The effects of the PG group on protein binding, cell internalization, protein function protection, and endo/lysosomal escape were systematically evaluated. Compared to the unmodified PAEs (L3), guanidyl rich PAEs (L3PG) presented superior efficiency of protein binding and protein internalization, mainly via clathrin-mediated endocytosis. In addition, both PAEs showed robust capabilities to deliver cytosolic proteins with different molecular weight (ranging from 30 to 464 kDa) and isoelectric points (ranging from 4.3 to 9), which were significantly improved in comparison with the commercial reagents of PULsin and Pierce Protein Transection Reagent. Moreover, L3PG successfully delivered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 ribonucleoprotein (RNP) into HeLa cells expressing green fluorescent protein (GFP) and achieved more than 80% GFP expression knockout. These results demonstrated that guanidyl modification on PAEs can enhance its capabilities for intracellular delivery of cytosolic functional proteins and CRISPR/Cas9 ribonucleoprotein. The guanidyl-rich PAEs are promising nonviral vectors for functional protein delivery and potential use in protein and nuclease-based gene editing therapies.

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