Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, Japan.
Biomacromolecules. 2011 Sep 12;12(9):3174-85. doi: 10.1021/bm2006714. Epub 2011 Aug 24.
Small interfering RNA (siRNA) has great therapeutic potential for the suppression of proteins associated with disease, but delivery methods are needed for improved efficacy. Here, we investigated the properties of micellar siRNA delivery vehicles prepared with poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) comprising lysine amines modified to contain amidine and thiol functionality. Lysine modification was achieved using 2-iminothiolane (2-IT) [yielding PEG-b-PLL(N2IM-IM)] or dimethyl 3,3'-dithiobispropionimidate (DTBP) [yielding PEG-b-PLL(MPA)], with modifications aimed to impart disulfide cross-linking ability without compromising cationic charge. These two lysine modification reagents resulted in vastly different chemistry contained in the reacted block copolymer, which affected micelle formation behavior and stability along with in vitro and in vivo performance. Amidines formed with 2-IT were unstable and rearranged into a noncharged ring structure lacking free thiol functionality, whereas amidines generated with DTBP were stable. Micelles formed with siRNA and PEG-b-PLL(N2IM-IM) at higher molar ratios of polymer/siRNA, while PEG-b-PLL(MPA) produced micelles only near stoichiometric molar ratios. In vitro gene silencing was highest for PEG-b-PLL(MPA)/siRNA micelles, which were also more sensitive to disruption under disulfide-reducing conditions. Blood circulation was most improved for PEG-b-PLL(N2IM-IM)/siRNA micelles, with a circulation half-life 3× longer than naked siRNA. Both micelle formulations are promising for siRNA delivery applications in vitro and in vivo.
小干扰 RNA（siRNA）在抑制与疾病相关的蛋白质方面具有巨大的治疗潜力，但需要改进的递送方法来提高疗效。在这里，我们研究了由聚乙二醇-嵌段-聚（L-赖氨酸）（PEG-b-PLL）制备的胶束 siRNA 递送载体的性质，该聚合物包含赖氨酸胺被修饰为含有亚胺和硫醇官能团。赖氨酸修饰是通过使用 2-亚氨基硫醇（2-IT）[得到 PEG-b-PLL（N2IM-IM）]或二甲基 3,3'-二硫代双丙酰亚胺（DTBP）[得到 PEG-b-PLL（MPA）]来实现的，修饰的目的是在不损害正电荷的情况下赋予二硫键交联能力。这两种赖氨酸修饰试剂导致反应的嵌段共聚物中包含的化学性质截然不同，这影响了胶束形成行为和稳定性以及体外和体内性能。用 2-IT 形成的亚胺不稳定，重排成缺乏游离硫醇官能团的非电荷环结构，而用 DTBP 生成的亚胺稳定。PEG-b-PLL（N2IM-IM）与 siRNA 形成胶束时的摩尔比更高，而 PEG-b-PLL（MPA）仅在接近化学计量摩尔比时形成胶束。PEG-b-PLL（MPA）/siRNA 胶束的体外基因沉默效果最高，而且在二硫键还原条件下更容易受到破坏。PEG-b-PLL（N2IM-IM）/siRNA 胶束的血液半衰期最长，延长了 3 倍，与裸 siRNA 相比，血液循环半衰期最长。这两种胶束制剂都有望在体外和体内用于 siRNA 递送应用。
