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.

Graphical Abstract

中文翻译：

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壳聚糖包被的PLGA纳米载体对siRNA和CRISPR / Cas9复合物细胞递送的效率

目的

大多数基于siRNA或CRISPR / Cas9系统的研究都依赖于病毒载体进行转染。然而，这些病毒载体是免疫原性的，这限制了它们在基因治疗中的应用。因此，需要鉴定具有更高安全性和改进靶向性的新型载体。因此，我们已经探索了可生物降解的，壳聚糖包被的PLGA纳米载体对于CRISPR / Cas9和siRNA的细胞内递送的潜力。在这项研究中，我们比较了壳聚糖包被的PLGA NPs（CS-PLGA NPs）与PLGA NPs的CRISPR和siRNA递送效率。

方法

这里介绍的是表面修饰的CS-PLGA NP和PLGA NP结合和递送siRNA和CRISPR / Cas9复合物的功效的制备和评估。

结果

载于PLGA NP上的Cy3 siRNA的内在化为4.6％，平均荧光强度（MFI）为13.76％，而CS PLGA的则为89％的内在化，MFI为67.95％。通过抗GFP siRNA和NP评估的体外GFP沉默导致PLGA NP沉默10-15％，而CS-PLGA NP沉默50-55％。CRISPR-Cas9质粒pX459与CS PLGA的GFP沉默率为80–83％，而PLGA的GFP沉默率为11％，商业脂质转染剂为13％。

结论

可生物降解的CS-PLGA NP对siRNA和CRISPRCas9表现出成功的负载和高结合效率，并导致有效的沉默。我们的研究报告称，CS-PLGA NPs可以成为有效递送siRNA和CRISPR / Cas9复合物的新型合适候选物。

图形概要