Nagaraj Harini, Jeon Taewon, Cicek Yagiz Anil, Goswami Ritabrita, Nasim Nourina, Mhaske Rukmini, Rotello Vincent M
Department of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, Amherst, MA, 01003, USA.
Molecular and Cellular Biology Graduate Program, University of Massachusetts Amherst, 230 Stockbridge Road, Amherst, MA, 01003, USA.
Pharm Res. 2025 Jun 23. doi: 10.1007/s11095-025-03884-6.
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.
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.
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.
Overall, this strategy signifies a highly transferrable and versatile method for effective storage of siRNA.
小干扰RNA（siRNA）可实现高度特异性和靶向性基因沉默，为一系列疾病提供了潜在的治疗方法。siRNA进入细胞质对于其发挥功效至关重要；当前的递送系统通常采用内吞摄取途径，由于逃逸效率低下导致siRNA降解。胍基功能化的聚（氧杂降冰片烯）酰亚胺（PONI）聚合物有助于将siRNA直接递送至细胞质，在体外和体内均具有出色的基因敲低效果。本文证明了使用冻干法生成稳定的粉末，这些粉末在重新溶解后仍保留出色的递送和敲低活性，为转化应用提供了关键工具。
将PONI-胍聚合物与siRNA混合形成PONI-胍/siRNA复合物。将生成的复合物冻干，并在不同温度条件下储存4周。重新溶解并递送后，通过共聚焦激光扫描显微镜评估siRNA进入细胞质的情况。使用流式细胞术在表达绿色荧光蛋白（GFP）的报告基因deGFP HEK 293 T细胞系中评估敲低效果。通过定量基因表达水平（qRT-PCR）和细胞生长抑制（alamar蓝分析法）评估重新溶解的PONI-胍/si_STAT3在4T1乳腺癌细胞中的功效。比较冻干复合物和新制备复合物的递送和治疗效率。
冻干复合物保留了新制备复合物的关键功能特征。重新悬浮的复合物促进了siRNA有效地递送至细胞质，并通过在4T1细胞中递送靶向信号转导和转录激活因子3（STAT-3）基因的siRNA显示出治疗相关性，成功抑制了细胞生长（约70%）并敲低了该基因（约80%）。
总体而言，该策略是一种高度可转移且通用的有效储存siRNA的方法。
