Cellular Uptake Mechanism of Nucleic Acid Nanocapsules and Their DNA-Surfactant Building Blocks.
Cellular Uptake Mechanism of Nucleic Acid Nanocapsules and Their DNA-Surfactant Building Blocks.
复制标题
核酸纳米胶囊及其 DNA 表面活性剂构件的细胞摄取机制。
DOI：
10.1021/acs.bioconjchem.3c00104
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发表时间：
2023
影响因子：
4.7
通讯作者：
Rouge,JessicaL
中科院分区：
文献类型：
--
作者：
Pal,Suman;delaFuente,InaF;Sawant,ShraddhaS;Cannata,JennaN;He,Wu;Rouge,JessicaL
核酸纳米胶囊（NAN）是酶响应性DNA官能化胶束，其被构建用于DNA-表面活性剂缀合物（DSC）的受控释放，所述DNA-表面活性剂缀合物呈现具有已证明的治疗潜力的序列。在此，我们研究了DSCs进入细胞内空间的机制，并确定了血清对NANs整体摄取和内化机制的影响。使用药理学抑制剂选择性地阻断某些途径，我们表明，通过共聚焦可视化的细胞分布和流式细胞术定量的总细胞协会，清道夫受体介导的，小窝依赖性的内吞作用是NAN的主要细胞摄取途径在存在和不存在血清。此外，由于NAN可以通过外部刺激（例如酶）触发以释放DSC，因此我们试图在基于细胞的测定之前检查由酶降解的颗粒的摄取概况。我们发现，虽然清道夫受体介导的，小窝依赖的内吞作用仍然在发挥作用，能量不依赖的途径，以及网格蛋白介导的内吞作用也参与。总的来说，这项研究有助于阐明包装到胶束NAN平台中的DSC的细胞溶质递送和治疗活性的早期步骤，同时揭示了DNA功能化纳米材料通常可以作为纳米结构和分子实体贩运到细胞中的方式。重要的是，我们的研究还表明，NAN设计特别是能够在血清存在下递送时稳定核酸，这是有效治疗性核酸递送的关键步骤。
Nucleic acid nanocapsules (NANs) are enzyme-responsive DNA-functionalized micelles built for the controlled release of DNA-surfactant conjugates (DSCs) that present sequences with demonstrated therapeutic potential. Here, we investigate the mechanisms by which DSCs gain access to intracellular spacein vitroand determine the effects of serum on the overall uptake and internalization mechanism of NANs. Using pharmacological inhibitors to selectively block certain pathways, we show, through confocal visualization of cellular distribution and flow cytometry quantification of total cellular association, that scavenger receptor-mediated, caveolae-dependent endocytosis is the major cellular uptake pathway of NANs in the presence and absence of serum. Furthermore, as NANs can be triggered to release DSCs by external stimuli such as enzymes, we sought to examine the uptake profile of particles degraded by enzymes prior to cell-based assays. We found that while scavenger receptor-mediated, caveolae-dependent endocytosis is still at play, energy-independent pathways as well as clathrin-mediated endocytosis are also involved. Overall, this study has helped to elucidate early steps in the cytosolic delivery and therapeutic activity of DSCs packaged into a micellular NAN platform while shedding light on the way in which DNA functionalized nanomaterials in general can be trafficked into cells both as nanostructures and as molecular entities. Importantly, our study also shows that the NAN design in particular is able to stabilize nucleic acids when delivered in the presence of serum, a critical step for effective therapeutic nucleic acid delivery.