Department of Medical Biotechnology, School of Advanced Technologies, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Medical Plants Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Sci Rep. 2021 Oct 15;11(1):20531. doi: 10.1038/s41598-021-00085-0.
Although siRNA is a promising technology for cancer gene therapy, effective cytoplasmic delivery has remained a significant challenge. In this paper, a potent siRNA transfer system with active targeting moieties toward cancer cells and a high loading capacity is introduced to inhibit drug resistance. Mesoporous silica nanoparticles are of great potential for developing targeted gene delivery. Amino-modified MSNs (NH-MSNs) were synthesized using a modified sol-gel method and characterized by FTIR, BET, TEM, SEM, X-ray diffraction, DLS, and H-NMR. MDR1-siRNA was loaded within NH-MSNs, and the resulting negative surface was capped by functionalized chitosan as a protective layer. Targeting moieties such as TAT and folate were anchored to chitosan via PEG-spacers. The loading capacity of siRNA and the protective effect of chitosan for siRNA were determined by gel retardation assay. MTT assay, flow cytometry, real-time PCR, and western blot were performed to study the cytotoxicity, cellular uptake assay, targeting evaluation, and MDR1 knockdown efficiency. The synthesized NH-MSNs had a particle size of ≈ 100 nm and pore size of ≈ 5 nm. siRNA was loaded into NH-MSNs with a high loading capacity of 20% w/w. Chitosan coating on the surface of siRNA-NH-MSNs significantly improved the siRNA protection against enzyme activity compared to naked siRNA-NH-MSNs. MSNs and modified MSNs did not exhibit significant cytotoxicity at therapeutic concentrations in the EPG85.257-RDB and HeLa-RDB lines. The folate-conjugated nanoparticles showed a cellular uptake of around two times higher in folate receptor-rich HeLa-RDB than EPG85.257-RDB cells. The chitosan-coated siRNA-NH2-MSNs produced decreased MDR1 transcript and protein levels in HeLa-RDB by 0.20 and 0.48-fold, respectively. The results demonstrated that functionalized chitosan-coated siRNA-MSNs could be a promising carrier for targeted cancer therapy. Folate-targeted nanoparticles were specifically harvested by folate receptor-rich HeLa-RDB and produced a chemosensitized phenotype of the multidrug-resistant cancer cells.
尽管 siRNA 是癌症基因治疗的一种很有前途的技术，但有效的细胞质递送仍然是一个重大挑战。在本文中，我们介绍了一种具有针对癌细胞的主动靶向部分和高载药能力的有效 siRNA 传递系统，以抑制耐药性。介孔硅纳米粒子在开发靶向基因传递方面具有很大的潜力。本文采用改进的溶胶-凝胶法合成了氨基修饰的 MSNs（NH-MSNs），并通过 FTIR、BET、TEM、SEM、X 射线衍射、DLS 和 H-NMR 进行了表征。MDR1-siRNA 被负载在 NH-MSNs 内，所得的负表面通过功能化壳聚糖作为保护层进行封闭。通过 PEG 间隔物将靶向部分（如 TAT 和叶酸）锚定到壳聚糖上。通过凝胶阻滞实验测定 siRNA 的载药量和壳聚糖对 siRNA 的保护作用。通过 MTT 测定、流式细胞术、实时 PCR 和 Western blot 研究细胞毒性、细胞摄取试验、靶向评价和 MDR1 基因敲低效率。合成的 NH-MSNs 的粒径约为 100nm，孔径约为 5nm。siRNA 以 20%w/w 的高载药量负载到 NH-MSNs 中。与裸 siRNA-NH-MSNs 相比，壳聚糖在 siRNA-NH-MSNs 表面的涂层显著提高了 siRNA 对酶活性的保护作用。在 EPG85.257-RDB 和 HeLa-RDB 细胞系中，治疗浓度的 MSNs 和修饰的 MSNs 没有表现出明显的细胞毒性。叶酸偶联的纳米颗粒在叶酸受体丰富的 HeLa-RDB 细胞中的细胞摄取量约为叶酸受体缺乏的 EPG85.257-RDB 细胞的两倍。壳聚糖包被的 siRNA-NH2-MSNs 使 HeLa-RDB 中的 MDR1 转录物和蛋白质水平分别降低了 0.20 倍和 0.48 倍。结果表明，功能化壳聚糖包被的 siRNA-MSNs 可能是一种有前途的靶向癌症治疗载体。叶酸靶向纳米颗粒被叶酸受体丰富的 HeLa-RDB 特异性摄取，并产生多药耐药癌细胞的化学增敏表型。
