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Purpose <p>siRNA enables highly specific and targeted gene silencing, offering potential treatment for a range of diseases. Cytosolic access of siRNA is essential for efficacy; Current delivery systems&#xa0;generally use endosomal uptake pathways, leading to siRNA degradation due to inefficient escape. Guanidinium functionalized poly(oxanorbornene)imide (PONI) polymers facilitate direct cytosolic siRNA delivery with excellent gene knockdown efficacy <i>in vitro</i> and <i>in vivo</i>. The use of lyophilization to generate stable powders that retain excellent delivery and knockdown activity when reconstituted is demonstrated, providing a key tool for translation.</p> Methods <p>PONI-Guan polymers were mixed with siRNA to form PONI-Guan/siRNA polyplexes. The generated polyplexes were lyophilized and stored at varying temperature conditions for a total duration of 4&#xa0;weeks. After reconstitution and delivery, cytosolic access of siRNA was assessed through confocal laser scanning microscopy. Knockdown efficacy was assessed in GFP expressing reporter deGFP HEK 293&#xa0;T cell line using flow cytometry. Efficacy of reconstituted PONI-Guan/si_STAT3 in 4T1 breast cancer cells was evaluated by quantifying gene expression levels (qRT-PCR) and cell growth inhibition (Alamar blue assay). Delivery and therapeutic efficiency were compared between lyophilized and freshly made polyplexes.</p> Results <p>Lyophilized polyplexes retained critical functional features of freshly made polyplexes. Resuspended polyplexes facilitated effective cytosolic delivery siRNA and showed therapeutic relevance through the delivery of siRNA targeting STAT-3 gene in 4T1 cells with successful cell growth inhibition (~ 70%) and knockdown (~ 80%) of the gene.</p> Conclusion <p>Overall, this strategy signifies a highly transferrable and versatile method for effective storage of siRNA.</p>

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Efficient Cytosolic Delivery of siRNA Using Lyophilized and Reconstituted Polymer-siRNA Polyplexes

  • 期刊论文

  • 发表日期：

    2025年6月23日

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  • Harini Nagaraj,
  • Taewon Jeon,
  • Yagiz Anil Cicek,
  • Ritabrita Goswami,
  • Nourina Nasim,
  • Rukmini Mhaske,
  • Vincent M. Rotello

摘要

Purpose

siRNA enables highly specific and targeted gene silencing, offering potential treatment for a range of diseases. Cytosolic access of siRNA is essential for efficacy; Current delivery systems generally use endosomal uptake pathways, leading to siRNA degradation due to inefficient escape. Guanidinium functionalized poly(oxanorbornene)imide (PONI) polymers facilitate direct cytosolic siRNA delivery with excellent gene knockdown efficacy in vitro and in vivo. The use of lyophilization to generate stable powders that retain excellent delivery and knockdown activity when reconstituted is demonstrated, providing a key tool for translation.

Methods

PONI-Guan polymers were mixed with siRNA to form PONI-Guan/siRNA polyplexes. The generated polyplexes were lyophilized and stored at varying temperature conditions for a total duration of 4 weeks. After reconstitution and delivery, cytosolic access of siRNA was assessed through confocal laser scanning microscopy. Knockdown efficacy was assessed in GFP expressing reporter deGFP HEK 293 T cell line using flow cytometry. Efficacy of reconstituted PONI-Guan/si_STAT3 in 4T1 breast cancer cells was evaluated by quantifying gene expression levels (qRT-PCR) and cell growth inhibition (Alamar blue assay). Delivery and therapeutic efficiency were compared between lyophilized and freshly made polyplexes.

Results

Lyophilized polyplexes retained critical functional features of freshly made polyplexes. Resuspended polyplexes facilitated effective cytosolic delivery siRNA and showed therapeutic relevance through the delivery of siRNA targeting STAT-3 gene in 4T1 cells with successful cell growth inhibition (~ 70%) and knockdown (~ 80%) of the gene.

Conclusion

Overall, this strategy signifies a highly transferrable and versatile method for effective storage of siRNA.