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.
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.