Most studies based on siRNA or CRISPR/Cas9 systems rely on viral vectors for their transfection. However, these viral vectors are immunogenic, which limits their application in gene therapy. Thus, identification of novel vectors with higher safety and improved targeting is desirable. Hence, we have explored the potential of biodegradable, chitosan coated PLGA nanocarriers for intracellular delivery of CRISPR/Cas9, and siRNA. In this investigation, we have compared the efficiency of chitosan-coated PLGA NPs (CS-PLGA NPs) with that of the PLGA NPs for the delivery of CRISPR and siRNA.
Presented here is the preparation and evaluation of specifically surface-modified CS-PLGA NPs and PLGA NPs on their efficacy for binding and delivery of siRNA and CRISPR/Cas9 complex.
Cy3 siRNA loaded on PLGA NPs showed an internalization of 4.6% and mean fluorescent intensity (MFI) of 13.76%, while that of CS PLGA resulted in 89% internalization and MFI of 67.95%. The in vitro GFP silencing assessed by anti-GFP siRNA and NPs resulted in 10–15% silencing by PLGA NPs and 50–55% silencing by CS-PLGA NPs. The GFP silencing by CRISPR-Cas9 plasmid pX459 with CS PLGA was 80–83%, while that of PLGA was 11% and of commercial Lipofectamine agent was 13%.
The biodegradable CS-PLGA NPs exhibited successful loading and high binding efficiency for siRNA as well as CRISPRCas9 and resulted in effective silencing. Our studies report that the CS-PLGA NPs can be a novel suitable candidate for the effective delivery of siRNA and CRISPR/Cas9 complex.
This is a preview of subscription content,to check access.
Access this article
Similar content being viewed by others
Gori JL, Hsu PD, Maeder ML, Shen S, Welstead GG, Bumcrot D. Delivery and specificity of CRISPR/Cas9 genome editing technologies for human gene therapy. Hum Gene Ther. 2015;26(7):443–51.
Xiao-Jie L, Hui-Ying X, Zun-Ping K, Jin-Lian C, Li-Juan J. CRISPR-Cas9: a new and promising player in gene therapy. J Med Genet. 2015;52(5):289–96.
Harrison MM, Jenkins BV, O’Connor-Giles KM, Wildonger J. A CRISPR view of development. Genes Dev. 2014;28(17):1859–72.
Wang F, Qi LS. Applications of CRISPR genome engineering in cell biology. Trends Cell Biol. 2016;26(11):875–88.
Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262–78.
Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2015;517(7536):583–8.
Lino CA, Harper JC, Carney JP, Timlin JA. Delivering CRISPR: a review of the challenges and approaches. Drug delivery. 2018;25(1):1234–57.
Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343(6166):84–7.
Han X, Liu Z, Jo M, Zhang K, Li Y, Zeng Z, et al. CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation. Sci Adv. 2015;1(7):e1500454.
Wang H-X, Li M, Lee CM, Chakraborty S, Kim HW, Bao G, et al. CRISPR/Cas9-based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery. Chem Rev. 2017;117(15):9874–906.
Tao, Y., et al., Application of nanoparticle-based siRNA and CRISPR/Cas9 delivery systems in gene-targeted therapy. 2019, Future Medicine.
Chen, F., M. Alphonse, and Q. Liu, Strategies for nonviral nanoparticle-based delivery of CRISPR/Cas9 therapeutics. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 2019: p. e1609.
Li L, He ZY, Wei XW, Gao GP, Wei YQ. Challenges in CRISPR/CAS9 delivery: potential roles of nonviral vectors. Hum Gene Ther. 2015;26(7):452–62.
Glass Z, Li Y, Xu Q. Nanoparticles for CRISPR–Cas9 delivery. Nature biomedical engineering. 2017;1(11):854–5.
Miller JB, Zhang S, Kos P, Xiong H, Zhou K, Perelman SS, et al. Non-viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA. Angew Chem Int Ed. 2017;56(4):1059–63.
Mout R, Ray M, Yesilbag Tonga G, Lee YW, Tay T, Sasaki K, et al. Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano. 2017;11(3):2452–8.
Glass Z, Lee M, Li Y, Xu Q. Engineering the delivery system for CRISPR-based genome editing. Trends Biotechnol. 2018;36(2):173–85.
Yang W, et al. CRISPR/Cas9: implications for modeling and therapy of neurodegenerative diseases. Front Mol Neurosci. 2016;9:30.
Liu C, Zhang L, Liu H, Cheng K. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J Control Release. 2017;266:17–26.
Bala, I., S. Hariharan, and M.R. Kumar, PLGA nanoparticles in drug delivery: the state of the art. Critical Reviews™ in Therapeutic Drug Carrier Systems, 2004. 21(5).
Astete CE, Sabliov CM. Synthesis and characterization of PLGA nanoparticles. J Biomater Sci Polym Ed. 2006;17(3):247–89.
Choi J-S, Seo K, Yoo J-W. Recent advances in PLGA particulate systems for drug delivery. Journal of Pharmaceutical Investigation. 2012;42(3):155–63.
Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers. 2011;3(3):1377–97.
Aghamiri S, Talaei S, Ghavidel AA, Zandsalimi F, Masoumi S, Hafshejani NH, et al. Nanoparticles-mediated CRISPR/Cas9 delivery: recent advances in cancer treatment. Journal of Drug Delivery Science and Technology. 2020;56:101533.
Chen F, Alphonse M, Liu Q. Strategies for nonviral nanoparticle-based delivery of CRISPR/Cas9 therapeutics. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 2020;12(3):e1609.
Liu, Qi, Wang, Chun, Zheng, Yadan, Zhao, Yu, Wang, Ying, Hao, Jialei, ... Kang, Chunsheng (2020). Virus-like nanoparticle as a co-delivery system to enhance efficacy of CRISPR/Cas9-based cancer immunotherapy. Biomaterials, 120275.
Wei T, Cheng Q, Min Y-L, Olson EN, Siegwart DJ. Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing. Nat Commun. 2020;11(1):1–12.
Zhang H, Bahamondez-Canas TF, Zhang Y, Leal J, Smyth HDC. PEGylated chitosan for nonviral aerosol and mucosal delivery of the CRISPR/Cas9 system in vitro. Mol Pharm. 2018;15(11):4814–26.
Chronopoulou L, Massimi M, Giardi MF, Cametti C, Devirgiliis LC, Dentini M, et al. Chitosan-coated PLGA nanoparticles: a sustained drug release strategy for cell cultures. Colloids Surf B: Biointerfaces. 2013;103:310–7.
Ragelle H, Riva R, Vandermeulen G, Naeye B, Pourcelle V, le Duff CS, et al. Chitosan nanoparticles for siRNA delivery: optimizing formulation to increase stability and efficiency. J Control Release. 2014;176:54–63.
The author is very thankful to Mr. Lalit Borade from TIFR, Mumbai, for giving the facility of TEM imaging. Mr. Anjan Ghosh has helped in acquiring the FACS data from BD FACS Melody. We also like to acknowledge Ms. Namrata, Ms. Soumya, and Mr. Lalit from the laboratory of Dr. Debjyoti, IGIB, New Delhi, for providing CRISPR/Cas9, plasmid, and HEK 293T GFP-positive cell sample.
The authors are thankful to Department of Science Technology (DST) Nanomission (SR/NM/NS1145/2012) and Indian Council of Medical Research (ICMR) (No. 3/1/3/JRF-2015/HRD-LS/29/31232/85) Government of India for the research grant and fellowship.
Conflict of Interest
The authors declare that they have no conflict to interest
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
About this article
Cite this article
Srivastav, A., Gupta, K., Chakraborty, D. et al. Efficiency of Chitosan-Coated PLGA Nanocarriers for Cellular Delivery of siRNA and CRISPR/Cas9 Complex. J Pharm Innov 17, 180–193 (2022). https://doi.org/10.1007/s12247-020-09496-4