De Rosa Giuseppe, Salzano Giuseppina
Department of Pharmacy, Università degli Studi di Napoli Federico II, Via Domenico Montesano 49, 80131, Naples, Italy,
Methods Mol Biol. 2015;1218:43-51. doi: 10.1007/978-1-4939-1538-5_4.
The therapeutic use of small interfering RNA (siRNA) represents a new and powerful approach to suppress the expression of pathologically genes. However, biopharmaceutical drawbacks, such as short half-life, poor cellular uptake, and unspecific distribution into the body, hamper the development of siRNA-based therapeutics. Poly(lactide-co-glycolide), (PLGA) microspheres can be a useful tool to overcome these issues. siRNA can be encapsulated into the PLGA microspheres, which protects the loaded nucleic acid against the enzymatic degradation. Moreover, PLGA microspheres can be injected directly into the action site, where the siRNA can be released in controlled manner, thus avoiding the need of frequent invasive administrations. The complete biodegradability of PLGA to monomers easily metabolized by the body, and its approval by FDA and EMA for parenteral administration, assure the safety of this copolymer and do not require the removal of the device after the complete drug release. In chapter, a basic protocol for the preparation of PLGA microspheres encapsulating siRNA is described. This protocol is based on a double emulsion/solvent evaporation technique, a well known and easy to reproduce method. This specific protocol has been developed to encapsulate a siRNA anti-TNFα in PLGA microspheres, and it has been designed and optimized to achieve high siRNA encapsulation efficiency and slow siRNA release in vitro. However, it can be extended also to other siRNA as well as other RNA or DNA-based oligonucleotides (miRNA, antisense, decoy, etc.). Depending on the applications, chemical modifications of the backbone and site-specific modification within the siRNA sequences could be required.
小干扰RNA（siRNA）的治疗用途代表了一种抑制病理基因表达的全新且强大的方法。然而，生物制药方面的缺点，如半衰期短、细胞摄取差以及在体内的非特异性分布，阻碍了基于siRNA的治疗药物的开发。聚（丙交酯-共-乙交酯）（PLGA）微球可能是克服这些问题的有用工具。siRNA可以被封装到PLGA微球中，这能保护负载的核酸不被酶降解。此外，PLGA微球可以直接注射到作用部位，在那里siRNA可以以可控的方式释放，从而避免频繁进行侵入性给药的需要。PLGA完全可生物降解为易于被身体代谢的单体，并且它已获得美国食品药品监督管理局（FDA）和欧洲药品管理局（EMA）的肠胃外给药批准，确保了这种共聚物的安全性，并且在药物完全释放后无需取出装置。在本章中，描述了制备包封siRNA的PLGA微球的基本方案。该方案基于双乳液/溶剂蒸发技术，这是一种众所周知且易于重复的方法。这个特定的方案是为了将一种抗TNFα的siRNA封装到PLGA微球中而开发的，并且它经过设计和优化以实现高siRNA包封效率和体外siRNA的缓慢释放。然而，它也可以扩展到其他siRNA以及其他基于RNA或DNA的寡核苷酸（miRNA、反义寡核苷酸、诱饵寡核苷酸等）。根据应用的不同，可能需要对siRNA序列的主链进行化学修饰和位点特异性修饰。
