School of Medicine, Chang Gung University, Kwei-San, Taoyuan 33302, Taiwan.
Department of Neurosurgery, Chang Gung Memorial Hospital, Linkou, Kwei-San, Taoyuan 33305, Taiwan.
Int J Mol Sci. 2020 Sep 26;21(19):7111. doi: 10.3390/ijms21197111.
In this study, we aimed to develop a multifunctional drug/gene delivery system for the treatment of glioblastoma multiforme by combining the ligand-mediated active targeting and the pH-triggered drug release features of graphene oxide (GO). Toward this end, we load irinotecan (CPT-11) to cetuximab (CET)-conjugated GO (GO-CET/CPT11) for pH-responsive drug release after endocytosis by epidermal growth factor receptor (EGFR) over-expressed U87 human glioblastoma cells. The ultimate injectable drug/gene delivery system was designed by co-entrapping stomatin-like protein 2 (SLP2) short hairpin RNA (shRNA) and GO-CET/CPT11 in thermosensitive chitosan--poly(-isopropylacrylamide) (CPN) polymer solution, which offers a hydrogel depot for localized, sustained delivery of the therapeutics after the in situ formation of CPN@GO-CET/CPT11@shRNA hydrogel. An optimal drug formulation was achieved by considering both the loading efficiency and loading content of CPT-11 on GO-CET. A sustained and controlled release behavior was found for CPT-11 and shRNA from CPN hydrogel. Confocal microscopy analysis confirmed the intracellular trafficking for the targeted delivery of CPT-11 through interactions of CET with EGFR on the U87 cell surface. The efficient transfection of U87 using SLP2 shRNA was achieved using CPN as a delivery milieu, possibly by the formation of shRNA/CPN polyplex after hydrogel degradation. In vitro cell culture experiments confirmed cell apoptosis induced by CPT-11 released from acid organelles in the cytoplasm by flow cytometry, as well as reduced SLP2 protein expression and inhibited cell migration due to gene silencing. Finally, in vivo therapeutic efficacy was demonstrated using the xenograft of U87 tumor-bearing nude mice through non-invasive intratumoral delivery of CPN@GO-CET/CPT11@shRNA by injection. Overall, we have demonstrated the novelty of this thermosensitive hydrogel to be an excellent depot for the co-delivery of anticancer drugs and siRNA. The in situ forming hydrogel will not only provide extended drug release but also combine the advantages offered by the chitosan-based copolymer structure for siRNA delivery to broaden treatment modalities in cancer therapy.
在这项研究中，我们旨在通过结合氧化石墨烯（GO）的配体介导的主动靶向和 pH 触发的药物释放特性，开发一种用于治疗多形性胶质母细胞瘤的多功能药物/基因传递系统。为此，我们将伊立替康（CPT-11）载入表皮生长因子受体（EGFR）过表达的 U87 人胶质母细胞瘤细胞内吞后具有 pH 响应性的 Cetuximab（CET）缀合的 GO（GO-CET/CPT11）中，用于药物释放。最终的可注射药物/基因传递系统是通过将 Stomatin-like 蛋白 2（SLP2）短发夹 RNA（shRNA）和 GO-CET/CPT11 共包封在温敏壳聚糖-聚（异丙基丙烯酰胺）（CPN）聚合物溶液中设计的，该溶液提供了一个水凝胶库，用于在原位形成 CPN@GO-CET/CPT11@shRNA 水凝胶后，对治疗剂进行局部、持续的递送。通过考虑 CPT-11 在 GO-CET 上的载药效率和载药量，实现了最佳的药物配方。CPN 水凝胶中发现 CPT-11 和 shRNA 具有持续和控制释放行为。共焦显微镜分析证实了靶向递送 CPT-11 通过 CET 与 U87 细胞表面上的 EGFR 相互作用的细胞内转运。使用 CPN 作为递送介质，可以有效地转染 U87 使用 SLP2 shRNA，可能是由于水凝胶降解后形成 shRNA/CPN 聚集体。体外细胞培养实验通过流式细胞术证实了 CPT-11 从细胞质酸性细胞器中释放引起的细胞凋亡，以及由于基因沉默导致 SLP2 蛋白表达减少和细胞迁移抑制。最后，通过向荷瘤裸鼠瘤内注射 CPN@GO-CET/CPT11@shRNA，通过非侵入性瘤内递送，在 U87 肿瘤裸鼠异种移植模型中证明了体内治疗效果。总体而言，我们证明了这种温敏水凝胶的新颖性，它是一种极好的用于共递送抗癌药物和 siRNA 的储库。原位形成的水凝胶不仅可以提供延长的药物释放，还可以结合壳聚糖基共聚物结构提供的用于 siRNA 递送的优势，从而拓宽癌症治疗的治疗方式。
