deRonde Brittany M, Torres Joe A, Minter Lisa M, Tew Gregory N
Department of Polymer Science and Engineering, University of Massachusetts Amherst , Amherst, Massachusetts 01003, United States.
Department of Veterinary and Animal Sciences, University of Massachusetts Amherst , Amherst, Massachusetts 01003, United States.
Biomacromolecules. 2015 Oct 12;16(10):3172-9. doi: 10.1021/acs.biomac.5b00795. Epub 2015 Sep 14.
RNA interference is gaining attention as a means to explore new molecular pathways and for its potential as a therapeutic; however, its application in immortal and primary T cells is limited due to challenges with efficient delivery in these cell types. Herein, we report the development of guanidinium-rich protein transduction domain mimics (PTDMs) based on a ring-opening metathesis polymerization scaffold that delivers siRNA into Jurkat T cells and human peripheral blood mononuclear cells (hPBMCs). Homopolymer and block copolymer PTDMs with varying numbers of guanidinium moieties were designed and tested to assess the effect cationic charge content and the addition of a segregated, hydrophobic block had on siRNA internalization and delivery. Internalization of fluorescently labeled siRNA into Jurkat T cells illustrates that the optimal cationic charge content, 40 charges per polymer, leads to higher efficiencies, with block copolymers outperforming their homopolymer counterparts. PTDMs also outperformed commercial reagents commonly used for siRNA delivery applications. Select PTDM candidates were further screened to assess the role the PTDM structure has on the delivery of biologically active siRNA into primary cells. Specifically, siRNA to hNOTCH1 was delivered to hPBMCs enabling 50-80% knockdown efficiencies, with longer PTDMs showing improved protein reduction. By evaluating the PTDM design parameters for siRNA delivery, more efficient PTDMs were discovered that improved delivery and gene (NOTCH) knockdown in T cells. Given the robust delivery of siRNA by these novel PTDMs, their development should aid in the exploration of T cell molecular pathways leading eventually to new therapeutics.
RNA干扰作为一种探索新分子途径的手段及其治疗潜力正受到关注；然而，由于在这些细胞类型中有效递送存在挑战，其在永生T细胞和原代T细胞中的应用受到限制。在此，我们报告了基于开环易位聚合支架开发的富含胍基的蛋白质转导结构域模拟物（PTDMs），该模拟物可将小干扰RNA（siRNA）递送至Jurkat T细胞和人外周血单核细胞（hPBMCs）。设计并测试了具有不同数量胍基部分的均聚物和嵌段共聚物PTDMs，以评估阳离子电荷含量以及添加隔离的疏水嵌段对siRNA内化和递送的影响。荧光标记的siRNA内化到Jurkat T细胞中表明，最佳阳离子电荷含量为每个聚合物40个电荷，可带来更高的效率，嵌段共聚物的表现优于其均聚物对应物。PTDMs也优于常用于siRNA递送应用的商业试剂。进一步筛选了选定的PTDM候选物，以评估PTDM结构在将生物活性siRNA递送至原代细胞中的作用。具体而言，针对人Notch1的siRNA被递送至hPBMCs，实现了50 - 80%的敲低效率，更长的PTDMs显示出更好的蛋白质减少效果。通过评估用于siRNA递送的PTDM设计参数，发现了更有效的PTDMs，其改善了T细胞中的递送和基因（Notch）敲低。鉴于这些新型PTDMs对siRNA的强大递送能力，它们的开发应有助于探索T细胞分子途径，最终带来新疗法。
