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

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.

On Complex Coacervate Core Micelles: Structure-Function Perspectives

“…Protein encapsulation with neutral-hydrophobic-charged triblock copolymers produces association colloids with higher stability. The hydrophobic block is less sensitive towards salt and pH variations and provides a barrier to protect the protein-rich coacervate [ 109 , 110 , 111 ]. Increasing the hydrophobicity of the charged block by the addition of carbon spacers between the polymer backbone and the charged side chains results in higher salt stability of protein-loaded C3Ms [ 109 ].…”

Assembly and Characterization of Polyelectrolyte Complex Micelles

“…The resulting self-assembled core-shell nanoparticles are variously referred to as polyelectrolyte complex micelles (PCMs), polyion complex micelles, block ionomer complexes, or coacervate-core micelles by analogy to surfactant micellization, even though all components of the system are hydrophilic 6,7 . PCMs' ability to encapsulate hydrophilic molecules such as proteins and nucleic acids, as well as the extensive tunability offered by the block copolymer carrier architecture makes them attractive candidates for delivering therapeutic molecules in vivo [8][9][10][11][12][13] .…”

Light: A Magical Tool for Controlled Drug Delivery

“…[ 155 ] Organic polymers offer a well‐documented pharmaceutically relevant platform that has been underexplored for photothermal drug encapsulation and delivery. [ 156 ] To date, different categories photothermal organic polymer nanoparticles for photothermal‐triggered drug delivery have been developed, including the amphiphilic polymer nanobioconjugates via coprecipitation of poly(cyclopentadithiophene‐ alt ‐diketopyrrolopyrrole) and photothermal modulator polystyrene‐ b ‐poly(acrylic acid) to regulate heat‐sensitive Ca 2+ ion channels in neurons, [ 157 ] semiconducting polymer L1057 nanoparticles fabricated by Pd‐catalyzed Stille polymerization of 6,6,12,12‐tetrakis(4‐hexylphenyl)‐ s ‐indacenodithieno[3,2‐ b ]thiophene‐bis(trimethylstannane) and 4,9‐dibromo‐6,7‐bis(4‐(hexyloxy)phenyl)[1,2,5]thiadiazolo[3,4‐ g ]quinoxaline for NIR‐II bioimaging‐guided photothermal cancer therapy, [ 158 ] the semiconducting copolymer poly[(diketopyrrolopyrrole‐ alt ‐cyclopentadithiophene)‐ ran ‐(diketopyrrolopyrrole‐alt‐thiadiazoloquinoxaline)] for improved anticancer hyperthermia treatment, [ 159 ] NIR‐absorbance polymer PDPP3T‐O14 composed of poly((2,5‐diyl‐2,3,5,6‐tetrahydro‐3, 6‐dioxo‐pyrrolo(3,4‐ c )pyrrole‐1,4‐diyl)‐ alt ‐(2,2′:5′,2″‐terthiophene‐5,5″‐diyl)) as backbone and oligo(ethylene glycol) as side chains for efficient disassembly of amyloid β (Aβ), [ 160 ] cyclo(Arg‐Gly‐Asp‐DPhe‐Lys(mpa))‐modified donor–acceptor structured conjugated polymer nanoagents polymerized via hydrophobic alkyl chain‐appended (4,8‐bis((2‐octyldodecyl)oxy)‐benzo[1,2‐b:4,5‐b′]dithiophene‐2,6‐diyl)bis(trimethylstannane)) and 4,8‐bis(5‐bromo‐4‐(2‐ethylhexyl)thiophen‐2‐yl)benzo[1,2‐c:4,5‐c′]bis[1,2,5]thiadiazole as the electron donor and acceptor for accurate spatiotemporal hyperthermia brain cancer treatment via scalp and skull, [ 161 ] nanodots via peptide‐tuned self‐assembly of light‐activable porphyrins for photoacoustic imaging and efficient cancer treatment, [ 94d ] porphyrin‐based covalent organic framework nanoplatform for photoacoustic imaging‐guided combined photothermal/photodynamic therapy. [ 162 ] The semiconducting polymer L1057 NPs acting as the theranostic nanosystems were effectively applied in NIR‐II imaging‐guided photothermal therapy.…”