Characterization and comparison of two novel nanosystems associated with siRNA for cellular therapy
Characterization and comparison of two novel nanosystems associated with siRNA for cellular therapy
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DOI：
10.1016/j.ijpharm.2015.11.020
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发表时间：
2016-01-30
影响因子：
5.8
通讯作者：
Montero-Menei, C. N.
中科院分区：
文献类型：
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作者：
Andre, E. M.;Pensado, A.;Montero-Menei, C. N.
为了指导干细胞的命运，通过小干扰RNA(SiRNA)精细地控制基因表达是一种新的、安全的、有前途的策略。通过这种方式，阻碍神经元承诺的蛋白质的表达可能被暂时抑制，从而推动分化。间充质干细胞(MSC)能分泌组织修复因子，具有免疫调节功能，可向神经元方向分化，是中枢神经系统细胞治疗研究的一种有前途的细胞来源。为了更好地驱动他们的神经元承诺，抑制因子-1沉默转录(REST)因子，可能会被siRNA技术抑制。设计能够安全输送核酸的新型纳米颗粒(NP)是成功开发这一战略的关键。在本研究中，我们开发并鉴定了两种不同的siRNA NP。一方面，设计了基于山梨醇单油酸酯(Span(R)80)的NP，其中含有阳离子组分聚L精氨酸或阳离子普鲁兰，从而允许小干扰RNA的结合。这些纳米粒子具有较小的尺寸(205 Nm)和负的表面电荷(-38 mV)。另一方面，脂类纳米胶囊(LNC)也被开发出来，使聚合物与脂类结合，并使siRNA与脂合物络合在一起。它们的尺寸为82 nm，表面正电荷为+7 mV。两种NP均可用适当的冷冻保护剂冷冻。通过检测REST基因的表达，检测不同剂量的siRNA对细胞的毒性和转染率。当与商品试剂推荐的250 ng/mLsiRNA-REST联合使用时，两种NP都能抑制约60%的REST表达。SPAN NP对人MSCs的毒性低于LNC，但尽管两种NP随着时间的推移对静息的抑制和对神经元连接的诱导相似，LNC-siREST诱导了更高的神经元标志的表达。因此，两种不同的siRNA NP被开发出来，为人类干细胞的分化提供了巨大的潜力，鼓励了在体外和体内进一步研究的追求。(C)2015爱思唯尔B.V.保留所有权利。
To direct stem cell fate, a delicate control of gene expression through small interference RNA (siRNA) is emerging as a new and safe promising strategy. In this way, the expression of proteins hindering neuronal commitment may be transiently inhibited thus driving differentiation. Mesenchymal stem cells (MSC), which secrete tissue repair factors, possess immunomodulatory properties and may differentiate towards the neuronal lineage, are a promising cell source for cell therapy studies in the central nervous system. To better drive their neuronal commitment the repressor Element-1 silencing transcription (REST) factor, may be inhibited by siRNA technology. The design of novel nanoparticles (NP) capable of safely delivering nucleic acids is crucial in order to successfully develop this strategy. In this study we developed and characterized two different siRNA NP. On one hand, sorbitan monooleate (Span (R) 80) based NP incorporating the cationic components poly-L-arginine or cationized pullulan, thus allowing the association of siRNA were designed. These NP presented a small size (205 nm) and a negative surface charge (-38 mV). On the other hand, lipid nanocapsules (LNC) associating polymers with lipids and allowing encapsulation of siRNA complexed with lipoplexes were also developed. Their size was of 82 nm with a positive surface charge of +7 mV. Both NP could be frozen with appropriate cryoprotectors. Cytotoxicity and transfection efficiency at different siRNA doses were monitored by evaluating REST expression. An inhibition of around 60% of REST expression was observed with both NP when associating 250 ng/mL of siRNA-REST, as recommended for commercial reagents. Span NP were less toxic for human MSCs than LNCs, but although both NP showed a similar inhibition of REST over time and the induction of neuronal commitment, LNC-siREST induced a higher expression of neuronal markers. Therefore, two different tailored siRNA NP offering great potential for human stem cell differentiation have been developed, encouraging the pursuit of further in vitro and in vivo in studies. (C) 2015 Elsevier B.V. All rights reserved.