Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China.
Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China.
Biomaterials. 2014 Sep;35(27):7978-91. doi: 10.1016/j.biomaterials.2014.05.068. Epub 2014 Jun 14.
Lack of safe and effective delivery vehicle is the main obstacle for siRNA mediated cancer therapy. In this study, we synthesized a pH-sensitive polymer of PEG grafted carboxymethyl chitosan (PEG-CMCS) and developed anionic-charged hybrid nanoparticles of PEG-CMCS and calcium phosphate (CaP) for siRNA delivery through a single-step self-assembly method in aqueous condition. The formed nanoparticles with charge of around -8.25 mv and average diameter of 102.1 nm exhibited efficient siRNA encapsulation and enhanced colloidal and serum stability. The test in vitro indicated that the nanoparticles entered into HepG2 cells by endocytosis, and achieved endosomal escape of siRNA effectively due to the pH-responsive disassembly of nanoparticles and dissolution of CaP in the endosome. Reporter gene silencing assay showed that luciferase siRNA delivered by the anionic nanoparticles could achieve gene silencing efficacy comparable to that of conventional Lipofectamine 2000. Additionally, dramatic hTERT knockdown mediated by the anionic nanoparticles transfection induced significant apoptosis of HepG2 cells in vitro. After intravenous injection in tumor-bearing BALB/c nude mice, the nanoparticles specifically accumulated into tumor regions by EPR effect, leading to efficient and specific gene silencing sequentially. Most importantly, the nanoparticles carrying hTERT siRNA inhibited tumor growth significantly via silencing hTERT expression and inducing cells apoptosis in HepG2 tumor xenograft. Moreover, comprehensive safety studies of the nanoparticles confirmed their superior safety both in vitro and in vivo. We concluded that the PEG-CMCS/CaP hybrid anionic nanoparticles possessed potential as a safe and effective siRNA delivery system for anticancer therapy.
缺乏安全有效的递送载体是 siRNA 介导的癌症治疗的主要障碍。在本研究中，我们合成了一种聚乙二醇接枝羧甲基壳聚糖（PEG-CMCS）的 pH 敏感聚合物，并通过一步自组装法在水相条件下开发了 PEG-CMCS 和磷酸钙（CaP）的阴离子带电混合纳米粒子用于 siRNA 递送。形成的纳米粒子带约-8.25 mV 的电荷，平均直径为 102.1nm，具有高效的 siRNA 包封和增强的胶体和血清稳定性。体外试验表明，纳米粒子通过内吞作用进入 HepG2 细胞，并由于纳米粒子的 pH 响应性解体和内体中 CaP 的溶解，有效地实现了 siRNA 的内体逃逸。报告基因沉默试验表明，由阴离子纳米粒子递送的荧光素酶 siRNA 可以实现与常规 Lipofectamine 2000 相当的基因沉默效果。此外，阴离子纳米粒子转染引起的 hTERT 明显敲低导致 HepG2 细胞在体外发生显著凋亡。在荷瘤 BALB/c 裸鼠静脉注射后，纳米粒子通过 EPR 效应特异性聚集到肿瘤区域，从而依次实现高效和特异性基因沉默。最重要的是，携带 hTERT siRNA 的纳米粒子通过沉默 hTERT 表达和诱导 HepG2 肿瘤异种移植物中的细胞凋亡，显著抑制肿瘤生长。此外，纳米粒子的综合安全性研究证实了它们在体外和体内的优越安全性。我们得出结论，PEG-CMCS/CaP 混合阴离子纳米粒子具有作为用于癌症治疗的安全有效的 siRNA 递送系统的潜力。
