Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Science for Life, Faculty of Science , Utrecht University , P.O. Box 80082, 3508 TB Utrecht , The Netherlands.
Laboratory of Materials and Interface Chemistry and Centre for Multiscale Electron Microscopy Department of Chemical Engineering and Chemistry , Eindhoven University of Technology , Eindhoven , 5600 MB , The Netherlands.
Biomacromolecules. 2018 Sep 10;19(9):3766-3775. doi: 10.1021/acs.biomac.8b00908. Epub 2018 Aug 24.
In this study, native chemical ligation (NCL) was used as a selective cross-linking method to form core-cross-linked thermosensitive polymeric micelles for drug delivery applications. To this end, two complementary ABA triblock copolymers having polyethylene glycol (PEG) as midblock were synthesized by atom transfer radical polymerization (ATRP). The thermosensitive poly isopropylacrylamide (PNIPAM) outer blocks of the polymers were copolymerized with either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys), P(NIPAM- co-HPMA-Cys)-PEG-P(NIPAM- co-HPMA-Cys) (PNC) or N-(2-hydroxypropyl)methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA), P(NIPAM- co-HPMA-ETSA)-PEG-P(NIPAM- co-HPMA-ETSA) (PNE). Mixing of these polymers in aqueous solution followed by heating to 50 °C resulted in the formation of thermosensitive flower-like micelles. Subsequently, native chemical ligation in the core of micelles resulted in stabilization of the micelles with a Z-average of 65 nm at body temperature. Decreasing the temperature to 10 °C only affected the size of the micelles (increased to 90 nm) but hardly affected the polydispersity index (PDI) and aggregation number ( N) confirming covalent stabilization of the micelles by NCL. CryoTEM images showed micelles with an uniform spherical shape and dark patches close to the corona of micelles were observed in the tomographic view. The dark patches represent more dense areas in the micelles which coincide with the higher content of HPMA-Cys/ETSA close to the PEG chain revealed by the polymerization kinetics study. Notably, this cross-linking method provides the possibility for conjugation of functional molecules either by using the thiol moieties still present after NCL or by simply adjusting the molar ratio between the polymers (resulting in excess cysteine or thioester moieties) during micelle formation. Furthermore, in vitro cell experiments demonstrated that fluorescently labeled micelles were successfully taken up by HeLa cells while cell viability remained high even at high micelle concentrations. These results demonstrate the potential of these micelles for drug delivery applications.
在这项研究中，采用天然化学连接（NCL）作为选择性交联方法，制备用于药物传递应用的核交联热敏聚合物胶束。为此，通过原子转移自由基聚合（ATRP）合成了两种具有聚乙二醇（PEG）作为中间嵌段的互补 ABA 三嵌段共聚物。聚合物的热敏聚异丙基丙烯酰胺（PNIPAM）外嵌段与 N-（2-羟丙基）甲基丙烯酰胺-半胱氨酸（HPMA-Cys）、P（NIPAM-co-HPMA-Cys）-PEG-P（NIPAM-co-HPMA-Cys）（PNC）或 N-（2-羟丙基）甲基丙烯酰胺-乙基硫代甘油酸琥珀酸（HPMA-ETSA）、P（NIPAM-co-HPMA-ETSA）-PEG-P（NIPAM-co-HPMA-ETSA）（PNE）共聚。将这些聚合物在水溶液中混合，然后加热至 50°C，形成热敏花状胶束。随后，在胶束的核中进行天然化学连接，导致在体温下形成具有 65nm Z 均粒径的稳定胶束。将温度降低至 10°C 仅影响胶束的大小（增加至 90nm），但几乎不影响多分散指数（PDI）和聚集数（N），证实 NCL 对胶束的共价稳定作用。冷冻透射电镜图像显示胶束具有均匀的球形形状，并且在断层视图中观察到靠近胶束冠的暗斑。暗斑代表胶束中更密集的区域，这与聚合动力学研究中发现的靠近 PEG 链的 HPMA-Cys/ETSA 含量较高相对应。值得注意的是，这种交联方法提供了通过使用 NCL 后仍然存在的巯基或通过简单调整胶束形成过程中聚合物之间的摩尔比（导致剩余的半胱氨酸或硫酯基）来连接功能分子的可能性。此外，体外细胞实验表明，荧光标记的胶束被 HeLa 细胞成功摄取，而即使在高胶束浓度下细胞活力仍然很高。这些结果表明这些胶束在药物传递应用中的潜力。
