Efficiency of Chitosan-Coated PLGA Nanocarriers for Cellular Delivery of siRNA and CRISPR/Cas9 Complex
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ORIGINAL ARTICLE
Efficiency of Chitosan-Coated PLGA Nanocarriers for Cellular Delivery of siRNA and CRISPR/Cas9 Complex Ashu Srivastav 1,2 & Kritika Gupta 1,2 & Debojyoti Chakraborty 3 & Prajakta Dandekar 2
&
Ratnesh Jain 1
Accepted: 10 September 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Purpose 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. Methods 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. Results 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%. Conclusions 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.
Keywords CS-PLGA nanoparticles . siRNA . CRISPR/Cas9 . Delivery . GFP
Introduction
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12247-020-09496-4) contains supplementary material, which is available to authorized users. * Prajakta Dandekar [email protected] * Ratnesh Jain [email protected] 1
Department of Chemical Engineering, Institute of Chemical Technology, NP Marg, Matunga E, Mumbai 400019, India
2
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, NP Marg, Matunga E, Mumbai 400019, India
3
RNA lab, CSIR-Institute of Genomics & Integrative Biology, Mathura Road, Delhi 110025, India
CRISPR/Cas9 is a potent gene-editing tool that has been employed to uncover the function of hundreds of genes [1]. Additionally, the simplicity and flexibility of CRISPR/Cas9 site-specific nuclease system has led to its widespread applications in various areas of biological research, such as identifying disease targets, discovering mechanisms of disease, development of model cell lines, transcriptional modulation, and development of transgene animals and pl
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