Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA.
Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
Methods Mol Biol. 2020;2105:261-281. doi: 10.1007/978-1-0716-0243-0_17.
Many important biological applications of peptide nucleic acids (PNAs) target nucleic acid binding in eukaryotic cells, which requires PNA translocation across at least one membrane barrier. The delivery challenge is further exacerbated for applications in whole organisms, where clearance mechanisms rapidly deplete and/or deactivate exogenous agents. We have demonstrated that nanoparticles (NPs) composed of biodegradable polymers can encapsulate and release PNAs (alone or with co-reagents) in amounts sufficient to mediate desired effects in vitro and in vivo without deleterious reactions in the recipient cell or organism. For example, poly(lactic-co-glycolic acid) (PLGA) NPs can encapsulate and deliver PNAs and accompanying reagents to mediate gene editing outcomes in cells and animals, or PNAs alone to target oncogenic drivers in cells and correct cancer phenotypes in animal models. In this chapter, we provide a primer on PNA-induced gene editing and microRNA targeting-the two PNA-based biotechnological applications where NPs have enhanced and/or enabled in vivo demonstrations-as well as an introduction to the PLGA material and detailed protocols for formulation and robust characterization of PNA/DNA-laden PLGA NPs.
许多肽核酸 (PNA) 的重要生物学应用都针对真核细胞中的核酸结合，这需要 PNA 穿过至少一个膜屏障进行转运。对于整个生物体中的应用，递药的挑战更加严峻，因为清除机制会迅速耗尽和/或使外源性试剂失活。我们已经证明，由可生物降解聚合物组成的纳米颗粒 (NP) 可以包裹并释放 PNA（单独或与共试剂一起），其数量足以在体外和体内介导所需的效果，而不会在受体细胞或生物体中产生有害反应。例如，聚（乳酸-共-乙醇酸）（PLGA）NP 可以包裹和输送 PNA 及伴随的试剂，以介导细胞和动物中的基因编辑结果，或单独输送 PNA 以靶向细胞中的致癌驱动基因并纠正动物模型中的癌症表型。在本章中，我们提供了关于 PNA 诱导的基因编辑和 microRNA 靶向的入门知识——这是两个已经通过 NP 增强和/或实现了体内演示的基于 PNA 的生物技术应用——以及 PLGA 材料的介绍和详细的制剂方案以及负载 PNA/DNA 的 PLGA NP 的稳健表征。
