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

IF 3.5 3区医学 Q2 CHEMISTRY, MULTIDISCIPLINARY

Pharmaceutical Research Pub Date : 2025-06-23 DOI:10.1007/s11095-025-03884-6

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

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引用次数: 0

Abstract

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.

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利用冻干和重组的聚合物-siRNA复合物高效的siRNA细胞质递送。

目的：siRNA能够实现高度特异性和靶向性的基因沉默，为一系列疾病提供潜在的治疗方法。siRNA的细胞质通路对疗效至关重要；目前的递送系统通常使用内体摄取途径，导致siRNA由于低效逃逸而降解。胍官能化聚（oxanorbornene）亚胺（PONI）聚合物在体外和体内具有良好的基因敲低效果，可促进siRNA的直接细胞质递送。使用冻干来产生稳定的粉末，在重组时保持良好的传递和击倒活性，为翻译提供了关键工具。方法：将PONI-Guan聚合物与siRNA混合形成PONI-Guan/siRNA多聚物。将生成的多聚体冻干并在不同温度条件下保存4周。重组和递送后，通过共聚焦激光扫描显微镜评估siRNA的细胞质通路。流式细胞术检测表达GFP报告细胞deGFP HEK 293 T细胞株的敲除效果。通过定量基因表达水平（qRT-PCR）和细胞生长抑制（Alamar蓝法）评估重组PONI-Guan/si_STAT3在4T1乳腺癌细胞中的作用。比较冻干和新鲜多聚物的递送和治疗效果。结果：冻干后的多聚物保留了新鲜多聚物的关键功能特征。重悬多聚体促进了siRNA的有效胞质递送，并通过在4T1细胞中递送靶向STAT-3基因的siRNA显示出治疗相关性，成功抑制了细胞生长（~ 70%）和敲低（~ 80%）该基因。结论：总的来说，这一策略意味着一种高度可转移和通用的siRNA有效储存方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Pharmaceutical Research 医学-化学综合

CiteScore

6.60

自引率

5.40%

发文量

276

审稿时长

3.4 months

期刊介绍： Pharmaceutical Research, an official journal of the American Association of Pharmaceutical Scientists, is committed to publishing novel research that is mechanism-based, hypothesis-driven and addresses significant issues in drug discovery, development and regulation. Current areas of interest include, but are not limited to: -(pre)formulation engineering and processing- computational biopharmaceutics- drug delivery and targeting- molecular biopharmaceutics and drug disposition (including cellular and molecular pharmacology)- pharmacokinetics, pharmacodynamics and pharmacogenetics. Research may involve nonclinical and clinical studies, and utilize both in vitro and in vivo approaches. Studies on small drug molecules, pharmaceutical solid materials (including biomaterials, polymers and nanoparticles) biotechnology products (including genes, peptides, proteins and vaccines), and genetically engineered cells are welcome.