Intracellular Delivery of His‐Tagged Genome‐Editing Proteins Enabled by Nitrilotriacetic Acid–Containing Lipidoid Nanoparticles

Abstract

and metal oxide nanoparticles, [7] etc. have been reported for protein delivery purposes. Our group has developed several combinatorial libraries of lipid-like molecule (lipidoid)-based nanoparticles (LNPs) for intracellular delivery applications [8] ; the effectiveness of the LNPs has been shown by us and others in both in vitro and in vivo studies. [8c,9] These cationic lipidoids contain amine head groups and the main driving force for cargo complexation is the electrostatic interaction between LNP carriers and anionic guest molecules (negatively charged proteins, mRNA, siRNA, pDNA, etc.). [8e,10] The possibility of using noncationic lipidoids and other types of supramolecular interactions for protein loading and delivery is explored in this study.One of the most widely used protein purification processes takes advantage of the coordination interaction between the nickel-immobilized beads and the imidazole groups on the His-tagged proteins. Dowdy [11] and Zuber [12] developed nitrilotriacetic acid (NTA) moiety-containing peptide transduction domains and synthetic polymers complexed with divalent nickel ions for His-tagged protein/nanoparticle cargo binding and intracellular delivery. Inspired by their designs, we report here the synthesis of three types of NTA-containing lipidoids with different hydrophobic tail structures (NTA-EC16, NTA-O16B, and NTA-O17O; Figure 1) for protein delivery. As a proof of concept, nanoparticles were fabricated using active lipidoids containing NTA groups, together with divalent nickel ions and helper lipids (cholesterol, phospholipid, and macromolecular lipids; Figure 1B) for optimized intracellular delivery efficiencies. Histagged proteins including green fluorescent protein (GFP), Cre recombinase variant, and recently developed CRISPR (clustered regularly interspaced short palindromic repeat)-associated protein 9 (Cas9) nuclease were complexed with the nanoparticle formulations and their internalization efficiencies as well as genome-editing activities of Cre recombinase and Cas9 ribonucleoprotein (RNP) were studied ( Figure 1A). Results and Discussion Lipidoid Synthesis and Nanoparticle FabricationNitrilotriacetic acid group-containing amphiphilic lipidoids NTA-EC16, NTA-O16B, and NTA-O17O ( Figure 1B) were Protein-and peptide-based therapeutics with high tolerance and specificity along with low off-target effects and genetic risks have attracted tremendous attention over the last three decades. Herein, a new type of noncationic lipidoid nanoparticle (LNP) is reported for His-tagged protein delivery. Active lipidoids are synthesized by conjugating a nitrilotriacetic acid group with hydrophobic tails (EC16, O16B, and O17O) and nanoparticles are formulated in the presence of nickel ions and helper lipids (cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). It is demonstrated that the newly developed LNPs are capable of delivering various His-tagged proteins including green fluore...

Protein and mRNA Delivery Enabled by Cholesteryl‐Based Biodegradable Lipidoid Nanoparticles

“…Next, Cas9 mRNAw as loaded into LNPs,a long with single-guide RNA( sgRNA) that targets as equence of the GFP gene.G FP-expressing HEK cells (GFP-HEK) were used in this study. [20] Successful Cas9 mRNAa nd sgRNAd elivery would result in ak nockout of GFP expression. Interestingly,none of the three tested lipids were capable of inducing ad istinct GFP knockout after 48 h (lipidoid/mRNA/sgRNA = 10/1/1;[ mRNA] = [sgRNA] = 0.86 mgmL À1 )( Figure S21C).…”

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

“…The results indicate that lipids with O17Se tails are more likely to form efficacious lipidoid nanoparticles (LNPs) for Cas9 RNP delivery. Besides cationic lipids, a library of noncationic ones were designed to deliver His-tagged proteins (Figure 5 E) 173 . The noncationic lipids were synthesized by conjugating a nitrilotriacetic acid (NTA) group onto a hydrophobic tail.…”

“…Non-cationic NTA-containing lipidoids for Cas9 RNP delivery. Red color identifying the leading amine heads or lipidoid for the intracellular delivery of Cas9 RNP 173 .…”

Intracellular Antibody Delivery Mediated by Lipids, Polymers, and Inorganic Nanomaterials for Therapeutic Applications

“…[40,41] Lipids are amphiphilic small molecules, either naturally occurring or synthetically constructed, which self-assemble into nanoparticles in selective solvents. The combinatorial library strategy has been particularly useful in synthesizing and identifying novel lipid formulations for small molecule drug, [42,43] protein, [44][45][46] and gene [47][48][49] deliveries. Many types of lipids have been tested for intracellular antibody delivery.…”

“…[155] As functional ligands and reactive groups can be readily incorporated into the terminal/side chains of CPDs, both covalent and noncovalent strategies were developed for cargo complexation. These strategies included those based off of avidin/biotin interaction, [156] His-tag/Ni(II)-NTA coordination, [46] and bioorthogonal conjugations. [157] Specifically, the disulfide bond on the Alexa Fluor 488-labeled goat antirabbit IgG was reduced to free thiols and modified with bis-sulfone and trans-cyclooctyne (TCO)-containing commercial reagent ThioLinker-TCO (Figure 2; thiol-based conjugation).…”