Development of Silica Nanoparticles as a Delivery System for Plasmid-Based Crispr/Cas9

Year 2022, Volume: 1 Issue: 1, 33 - 39, 31.12.2022

Gozde Ultav , Kubra Mac Sena Kizilboga Vedat Gundogdu Hayrettin Tonbul , Emine Şalva

Abstract

Clustered regular interspace short palindromic repeat (CRISPR)/CRISPR-associated system (Cas) is
a promising technology for gene editing systems and genome manipulation. Transferring the CRISPR vector to cells
is an important aspect of the effective use of this technology. In this study, we aimed to develop a new delivery
system using silica nanoparticles (SNPs) with the CRISPR cas9 vector. SNPs were synthesized by the Stöber method.
The synthesized nanoparticles were analyzed with the Dynamic Light Scattering (DLS) method and approximately
100 nm SNPs were obtained. EF1a-GFP CRISPR/Cas9 plasmid has been transfected to the Escherichia coli (E.coli)
DH5α and isolated from the strain using the plasmid DNA isolation Kit. The isolated pCas-EF1a-GFP CRISPR/Cas9
plasmid was imaged by agarose gel electrophoresis. CRISPR/Cas9 plasmid (pCRISPR) attached to SNP by
electrostatic interactions and obtained pCRISPR/SNP complexes were checked by agarose gel electrophoresis.
Results show average particle size and zeta potential of obtained pCRISPR/SNP nanoparticles were among 146.6-
272.7 nm and -20.2 - +16,9 mV, respectively and full complexation was achieved at 1/10 pCRISPR/SNP w/w ratio.
Consequently, optimized silica nanoparticles can be a good candidate for the delivery of CRISPR/Cas9 plasmid.

Keywords

CRISPR/Cas9, plasmid, silica nanoparticle, delivery system, zeta potential, particle size.

References

• [1] Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nature Biotechnology, 2020; 38(7): 824-844.
• [2] CRISPR’s breakthrough problem. C&EN Global Enterprise, 2017. 95(7): 28-33.
• [3] New CRISPR inhibitors found. C&EN Global Enterprise, 2018; 96(37): 6-6.
• [4] Lino CA et al. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv, 2018; 25(1): 1234-1257.
• [5] Yip BH. Recent Advances in CRISPR/Cas9 Delivery Strategies. Biomolecules, 2020; 10(6).
• [6] Kretzmann JA et al. Targeted Therapeutic Genome Engineering: Opportunities and Bottlenecks in Medical Translation, in Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity. 2019, American Chemical Society. 1-34.
• [7] Wang HX et al. CRISPR/Cas9-Based Genome Editing for Disease Modeling and Therapy: Challenges and Opportunities for Nonviral Delivery. Chemical Reviews, 2017; 117(15): 9874-9906.
• [8] Li L, Hu S, Chen X. Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities. Biomaterials, 2018; 171: 207-218.
• [9] Buck J et al. Lipid-Based DNA Therapeutics: Hallmarks of Non-Viral Gene Delivery. ACS Nano, 2019: 13(4); 3754-3782.
• [10] Patra JK et al. Nano based drug delivery systems: recent developments and future prospects. Journal of Nanobiotechnology, 2018; 16(1): 71.
• [11] Suk JS et al PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Advanced Drug Delivery Reviews, 2016; 99: 28-51.
• [12] Selvarajan V, Obuobi S, Ee PLR. Silica Nanoparticles—A Versatile Tool for the Treatment of Bacterial Infections. 2020; 8.
• [13] Ultav G et al. Silica nanoparticle synthesis by experimental design for drug and gene delivery applications. Journal of Research in Pharmacy, 2022; 27(1): 12-22.
• [14] Chen X et al. A convenient and rapid method for genetic transformation of E. coli Anat. J. Pharm. Sci 2022:1(1) with plasmids. Antonie Van Leeuwenhoek, 2001; 80(3-4): 297-300.
• [15] Ultav G et al. pH-sensitive chitosanPEG-decorated hollow mesoporous silica nanoparticles could be an effective treatment for acute myeloid leukemia (AML). Journal of Nanoparticle Research, 2022; 24(2): 40.
• [16] Ultav G, Tonbul H, Salva E. An effective VEGF-siRNA delivery via folic acid decorated and pegylated silica nanoparticles. Journal of Drug Delivery Science and Technology, 2022; 76: 103828.