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 deliv...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-01-01
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| Series: | Journal of Nanobiotechnology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12951-019-0564-1 |
| _version_ | 1798026352039821312 |
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| author | Ami Jo Veronica M. Ringel-Scaia Dylan K. McDaniel Cassidy A. Thomas Rui Zhang Judy S. Riffle Irving C. Allen Richey M. Davis |
| author_facet | Ami Jo Veronica M. Ringel-Scaia Dylan K. McDaniel Cassidy A. Thomas Rui Zhang Judy S. Riffle Irving C. Allen Richey M. Davis |
| author_sort | Ami Jo |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T18:35:08Z |
| format | Article |
| id | doaj.art-2acf97cb89c5446d8bc388d89a413f52 |
| institution | Directory Open Access Journal |
| issn | 1477-3155 |
| language | English |
| last_indexed | 2024-04-11T18:35:08Z |
| publishDate | 2020-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Nanobiotechnology |
| spelling | doaj.art-2acf97cb89c5446d8bc388d89a413f522022-12-22T04:09:19ZengBMCJournal of Nanobiotechnology1477-31552020-01-0118111410.1186/s12951-019-0564-1Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR–Cas9 plasmidAmi Jo0Veronica M. Ringel-Scaia1Dylan K. McDaniel2Cassidy A. Thomas3Rui Zhang4Judy S. Riffle5Irving C. Allen6Richey M. Davis7Department of Chemical Engineering, Virginia TechGraduate Program in Translational Biology, Medicine, and Health, Virginia TechDepartment of Biomedical Sciences & Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia TechDepartment of Biomedical Sciences & Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia TechDepartment of Chemistry, Virginia TechDepartment of Chemistry, Virginia TechGraduate Program in Translational Biology, Medicine, and Health, Virginia TechDepartment of Chemical Engineering, Virginia TechAbstract 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.https://doi.org/10.1186/s12951-019-0564-1NanoprecipitationTransfectionCRISPR–Cas9PLGA nanoparticles |
| spellingShingle | Ami Jo Veronica M. Ringel-Scaia Dylan K. McDaniel Cassidy A. Thomas Rui Zhang Judy S. Riffle Irving C. Allen Richey M. Davis Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR–Cas9 plasmid Journal of Nanobiotechnology Nanoprecipitation Transfection CRISPR–Cas9 PLGA nanoparticles |
| title | Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR–Cas9 plasmid |
| title_full | Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR–Cas9 plasmid |
| title_fullStr | Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR–Cas9 plasmid |
| title_full_unstemmed | Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR–Cas9 plasmid |
| title_short | Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR–Cas9 plasmid |
| title_sort | fabrication and characterization of plga nanoparticles encapsulating large crispr cas9 plasmid |
| topic | Nanoprecipitation Transfection CRISPR–Cas9 PLGA nanoparticles |
| url | https://doi.org/10.1186/s12951-019-0564-1 |
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