Block Polymer Micelles Enable CRISPR/Cas9 Ribonucleoprotein Delivery: Physicochemical Properties Affect Packaging Mechanisms and Gene Editing Efficiency

Zhe Tan, Yaming Jiang, Mitra S. Ganewatta, Ramya Kumar, Allison Keith, Kirk Twaroski, Thomas Pengo, Jakub Tolar, Timothy P. Lodge, Theresa M. Reineke

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

Abstract

Gene editing with CRISPR/Cas9 is revolutionizing biotechnology and medical research, yet affordable, efficient, and tailorable delivery systems are urgently needed to advance translation. Herein, a series of monodisperse amphiphilic block polymers poly[ethylene oxide-b-2-(dimethylamino) ethyl methacrylate-b-n-butyl methacrylate] (PEO-b-PDMAEMA-b-PnBMA) that housed three PEO lengths (2, 5, and 10 kDa) and a variant lacking PEO (PDMAEMA-b-PnBMA) were synthesized via controlled radical polymerization and assembled into well-defined spherical cationic micelles. The cationic micelles were complexed via electrostatic interactions with Cas9 protein/guide RNA ribonucleoproteins (RNPs) that exhibit anionic charges due to the overhanging RNA. The resulting micelleplex formulations in both phosphate-buffered saline (PBS) and water were screened via high content analysis for gene editing efficiency. The micelle variant with the 10 kDa PEO block offered the highest gene editing performance and was advanced for in-depth characterization. For the first time, quantitative static and dynamic light scattering characterization and cryogenic transmission electron microscopy images of Cas9 protein/guideRNA RNP loading into well-defined micelleplex nanoparticles are revealed, where the formulation solvent was found to play a major role in the physicochemical properties and biological performance. In PBS, the solutions containing the micelles (63 triblock polymers per micelle) were assembled with the Cas9 protein/guideRNA RNP payloads offering uniform loading of 14 RNPs per micelleplex and moderate editing efficiency; this homogeneous system offers promise for future in vivo/preclinical applications. Interestingly, when the uniform micelles were formulated with the RNP payloads in water, larger multimicelleplex nanoparticles were formed that offered double the editing efficiency of Lipofectamine 2000 (40% gene editing) due to the rapid sedimentation kinetics of the larger colloids onto adherent cells, offering promising in vitro, ex vivo, and/or cell therapy applications. This work presents the first quantitative demonstration of tailorable block polymer micelle formulations for advancing CRISPR/Cas9 RNP delivery and fundamental correlation of the solutions physics to biological performance.

Original languageEnglish (US)
Pages (from-to)8197-8206
Number of pages10
JournalMacromolecules
Volume52
Issue number21
DOIs
StatePublished - Nov 12 2019

Bibliographical note

Funding Information:
This work was supported partially by the Defense Advanced Research Projects Agency (DARPA) under contract number N660011824041 and the National Science Foundation through the University of Minnesota MRSEC under award number DMR-1420013. Parts of this work was carried out in the Characterization Facility, University of Minnesota, which has received capital equipment funding from the NSF through the UMN MRSEC program under award number DMR-1420013. The image cytometry experiment was done with the assistance of Dr. Guillermo Marques at the University of Minnesota - University Imaging Centers, http://uic.umn.edu . We thank Lisa Zeeb for her help with graphic design and generation.

Publisher Copyright:
Copyright © 2019 American Chemical Society.

How much support was provided by MRSEC?

  • Partial

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