Sun Di, Okosun Blessing O, Xue Yujie, Tayutivutikul Kirati, Smith Kaitlyn H, Darland Diane C, Zhao Julia X
Department of Chemistry, University of North Dakota, Grand Forks, ND, 58202, USA.
Department of Biology, University of North Dakota, Grand Forks, ND, 58202, USA.
J Mater Chem B. 2025 Feb 5;13(6):2124-2139. doi: 10.1039/d4tb01954g.
Regulation of gene expression in eukaryotic cells is critical for cell survival, proliferation, and cell fate determination. Misregulation of gene expression can have substantial, negative consequences that result in disease or tissue dysfunction that can be targeted for therapeutic intervention. Several strategies to inhibit gene expression at the level of mRNA transcription and translation have been developed, such as anti-sense inhibition and CRISPR-Cas9 gene editing. However, these strategies have some limitations in terms of specificity, toxicity, and ease of use. We have designed a nanomaterials-based tool to inhibit gene expression in eukaryotic cells with a potential application in basic and biomedical research. At the heart of our rational design approach is a polymer dots (Pdots)-based nanoplatform that can provide a means to deliver gene-specific small interfering (siRNA) into cells while at the same time providing a visualization mechanism to determine which cells have taken up the siRNA. The Pdots that we designed and synthesized had an average size 64.25 ± 0.60 nm and a zeta potential that was +37.40 ± 8.28 mV. The Pdot-1 nmole Gapdh siRNA showed an average size of 82.27 ± 9.83 nm, with the zeta potential values determined to be -52.00 ± 6.05 mV in the HEPES buffer. Both Pdots and Pdot-siRNA displayed two emission peaks in the visible (588 nm) and near-infrared (NIR) emission range (775 nm). We treated primary cultures of mouse brain-derived microvascular cells with Pdot-Gapdh siRNA and observed uniform cellular uptake of the nanomaterial in the cells and reduced intensity of Gapdh immunolabeling. Our results highlight the potential application of Pdot-siRNA for gene expression targeting with simultaneous visual monitoring of Pdot-siRNA delivery. The simple design offers a flexible and novel strategy to inhibit a wide range of mRNA targets with minimal toxicity, high efficiency, and focused cell visualization.
真核细胞中基因表达的调控对于细胞存活、增殖及细胞命运决定至关重要。基因表达失调会产生严重的负面后果，导致疾病或组织功能障碍，而这可成为治疗干预的靶点。目前已开发出多种在mRNA转录和翻译水平抑制基因表达的策略，如反义抑制和CRISPR-Cas9基因编辑。然而，这些策略在特异性、毒性和易用性方面存在一些局限性。我们设计了一种基于纳米材料的工具，用于抑制真核细胞中的基因表达，在基础研究和生物医学研究中具有潜在应用价值。我们合理设计方法的核心是一种基于聚合物点（Pdots）的纳米平台，它既能提供将基因特异性小干扰RNA（siRNA）递送至细胞的手段，又能提供一种可视化机制来确定哪些细胞摄取了siRNA。我们设计并合成的Pdots平均尺寸为64.25±0.60 nm，zeta电位为+37.40±8.28 mV。在HEPES缓冲液中，1 nmol Pdots与GAPDH siRNA复合物的平均尺寸为82.27±9.83 nm，zeta电位值为-52.00±6.05 mV。Pdots和Pdot-siRNA在可见光（588 nm）和近红外（NIR）发射范围（775 nm）均显示出两个发射峰。我们用Pdot-Gapdh siRNA处理小鼠脑源微血管细胞的原代培养物，观察到纳米材料在细胞中被均匀摄取，且Gapdh免疫标记强度降低。我们研究结果凸显了Pdot-siRNA在基因表达靶向及Pdot-siRNA递送同步可视化监测方面的潜在应用价值。这种简单的设计提供了一种灵活且新颖的策略，能够以最小的毒性、高效率及聚焦的细胞可视化来抑制多种mRNA靶点。
