Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR–Cas9 plasmid

Abstract

Background: The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy. Despite promising reports of the utility of CRISPR-Cas9 for in vivo gene editing, a principal problem in implementing this new process is delivery of high molecular weight DNA into cells. Results: Using poly(lactic-co-glycolic acid) (PLGA), a nanoparticle carrier was designed to deliver a model CRISPR-Cas9 plasmid into primary bone marrow derived macrophages. The engineered PLGA-based carriers were approximately 160 nm and fluorescently labeled by encapsulation of the fluorophore 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene). An amine-end capped PLGA encapsulated 1.6 wt% DNA, with an encapsulation efficiency of 80%. Release studies revealed that most of the DNA was released within the first 24 h and corresponded to ~ 2-3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24 h of treatment with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells. Conclusions: In this work, plasmids for the CRISPR-Cas9 system were encapsulated in nanoparticles comprised of PLGA and were shown to induce expression of bacterial Cas9 in murine bone marrow derived macrophages in vitro. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR-Cas9 system.

Encapsulation of Large-Size Plasmids in PLGA Nanoparticles for Gene Editing: Comparison of Three Different Synthesis Methods

“…These results could also be explained by electrostatic interactions since PBS contains salts that create a different ionic environment from that of water, which could change the interplay between pDNA and PLGA. Indeed, these data are consistent with the literature, in which other authors have reported up to 50% pDNA release from PLGA nanoparticles diluted in PBS at pHs ranging from 4.5 to 7 during the first hours after synthesis [ 45 , 46 ].…”

“…Alternatively, nanoprecipitation (or the solvent displacement method) relies on the mixture of organic and aqueous miscible solvents, taking advantage of the polymer’s low solubility in one of those solvents to induce precipitation of the polymer into nanoparticles upon evaporation of the organic solvent, typically by stirring or by reduced pressure. Although this method was originally designed for hydrophobic compound entrapment [ 44 ], it has been recently modified for hydrophilic compounds, such as DNA [ 45 , 46 ].…”

In vivo gene delivery mediated by non-viral vectors for cancer therapy

“…Also, the risk of the integration of plasmid into the genomic materials may reduce their potential for wide clinical applications. However, several non-viral delivery systems have been introduced for the efficient transfer of plasmid-based CRISPR/Cas9 systems including dendrimers, polymers, polypeptides and polysaccharides such as PLGA as well as lipid encapsulated gold NPs, PEI magnetic NPs and multifunctional nucleus-targeting core-shell artificial viruses [336][337][338][339][340][341].…”

Physical and mechanical cues affecting biomaterial-mediated plasmid DNA delivery: insights into non-viral delivery systems

“…Porous material may also result in more biodegradable, than non-porous ones [ 99 , 101 ]. Different DNA encapsulation techniques on oligo (polyethylene glycol) fumarate (OPF) hydrogels [ 103 ], alginate [ 104 ], polyethylene glycol ( PEG) [ 105 , 106 ], hyaluronic acid [ 99 ], fibrin [ 107 ], PLGA [ 23 , 108 ] or gelatin [ 109 ] hydrogels were described. Similarly, immobilisation on polyacrylamide [ 110 ], cationised gelatin hydrogels [ 111 ], poly(beta-amino ester)(PBE)/PLGA microparticles [ 112 ] and PLGA biomaterials [ 113 ], on polydopamine (PD) conjugated materials [ 114 ] and of collagen-mimetic peptides – conjugated polyplexes [ 115 ] were performed.…”