(473f) Engineering PLGA Nanoparticles to Encapsulate Large CRISPR-Cas9 Plasmids
Previous gene transfection approaches have used both chemical and physical stimuli, but these often destabilize cellular membranes resulting in unacceptable cytotoxicity especially in test animals.1 We hypothesize that using polymer nanoparticles will protect the plasmid from degradation en route and increase delivery into the cell without the harmful side effects seen using other methods for transfection.
Using a modified nanoprecipitation method2, a poly(lactic-co-glycolic acid) (PLGA) nanoparticle carrier with a diameter ~ 160 nm was engineered to deliver a CRISPR-Cas9 plasmid designed to correct a single point mutation in the Tlr4 gene in mice that cause them to not react to endotoxins, such as lipopolysaccharides (LPS). The PLGA carrier was also fluorescently labeled by encapsulation of a commercially available fluorophore, 6,13-Bis(triisopropylsilylethynl) pentacene (TIPS pentacene), that has not been used in the biomedical field until recently.3
However, before even getting to animal studies, the particles must first be appropriately designed, fabricated, and characterized to ensure that the requirements of the application are met. The focus of the research to be presented is on the particle processing and engineering that precedes the cell and animal studies. This work addresses the questions: What is the best solvent system to use for particle fabrication? What are the resulting sizes and zeta potentials? What is the composition of the polymeric carrier as well as what was successfully loaded? How can the particles be processed for long-term storage stability? Once these are answered, then the method can be optimized to increase DNA loading. An amine-end capped PLGA was used to form nanoparticles loaded with 1.6 wt% DNA, corresponding to an encapsulation efficiency of 80%. This loading is comparable to the ~1 wt% loadings seen for plasmids half the size. In release studies, most of the DNA was released within the first 24 hours and corresponded to ~2-3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24 hours of incubation with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells. These results indicate that the process described here will be effective for future in vivo applications of the CRISPR-Cas9 system.
- Bauer, M.; Kristensen, B. W.; Meyer, M.; Gasser, T.; Widmer, H. R.; Zimmer, J.; Ueffing, M., Toxic effects of lipid-mediated gene transfer in ventral mesencephalic explant cultures. Basic Clin Pharmacol 2006, 98 (4), 395-400.
- Niu, X. M.; Zou, W. W.; Liu, C. X.; Zhang, N.; Fu, C. H., Modified nanoprecipitation method to fabricate DNA-loaded PLGA nanoparticles. Drug Dev Ind Pharm 2009, 35 (11), 1375-1383.
- McDaniel, D. K.; Jo, A.; Ringel-Scaia, V. M.; Coutermarsh-Ott, S.; Rothschild, D. E.; Powell, M. D.; Zhang, R.; Long, T. E.; Oestreich, K. J.; Riffle, J. S.; Davis, R. M.; Allen, I. C., TIPS pentacene loaded PEO-PDLLA core-shell nanoparticles have similar cellular uptake dynamics in M1 and M2 macrophages and in corresponding in vivo microenvironments. Nanomedicine: Nanotechnology, Biology and Medicine 2017, 13 (3), 1255-1266.