Lechanteur Anna, Sanna Vincent, Duchemin Amandine, Evrard Brigitte, Mottet Denis, Piel Géraldine
Laboratory of Pharmaceutical Technology and Biopharmacy, CIRM, University of Liège, CHU Bat B36 Tour 4, +2, 1 avenue de l'Hopital, 4000 Liège, Belgium.
Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases, University of Liège, 4000 Liège, Belgium.
Nanomaterials (Basel). 2018 Apr 24;8(5):270. doi: 10.3390/nano8050270.
In recent year, cationic liposomes have gained a lot of attention for siRNA delivery. Despite this, intracellular barriers as endosomal escape and cytosolic delivery of siRNA still represent a challeng, as well as the cytotoxicity due to cationic lipids. To address these issues, we developed four liposomal formulations, composed of two different cationic lipids (DOTAP and DC-Cholesterol) and different ratio of co-lipids (cholesterol and DOPE). The objective is to dissect these impacts on siRNA efficacy and cytotoxicity. Liposomes were complexed to siRNA at six different N/P molar ratios, physico-chemical properties were characterized, and consequently, N/P 2.5, 5 and 10 were selected for in vitro experiments. We have shown that cytotoxicity is influenced by the N/P ratio, the concentration of cationic lipid, as well as the nature of the cationic lipid. For instance, cell viability decreased by 70% with liposomes composed of DOTAP/Cholesterol/DOPE 1/0.75/0.5 at a N/P ratio 10, whereas the same formulation at a N/P ratio of 2.5 was safe. Interestingly, we have observed differences in terms of mRNA knock-down efficiency, whereas the transfection rate was quite similar for each formulation. Liposomes containing 50% of DOPE induced a mRNA silencing of around 80%. This study allowed us to highlight crucial parameters in order to develop lipoplexes which are safe, and which induce an efficient intracytoplasmic release of siRNA.
近年来，阳离子脂质体在用于小干扰RNA（siRNA）递送方面备受关注。尽管如此，细胞内屏障如内体逃逸和siRNA的胞质递送仍然是一个挑战，阳离子脂质引起的细胞毒性也是如此。为了解决这些问题，我们开发了四种脂质体制剂，它们由两种不同的阳离子脂质（二油酰基磷脂酰乙醇胺-聚乙二醇-胆固醇[DOTAP]和二油酰基胆固醇[DC-胆固醇]）以及不同比例的共脂质（胆固醇和二油酰基磷脂酰乙醇胺[DOPE]）组成。目的是剖析这些因素对siRNA功效和细胞毒性的影响。脂质体与siRNA以六种不同的氮/磷（N/P）摩尔比进行复合，对其物理化学性质进行了表征，随后选择N/P 2.5、5和10用于体外实验。我们已经表明，细胞毒性受N/P比、阳离子脂质浓度以及阳离子脂质的性质影响。例如，由DOTAP/胆固醇/DOPE 1/0.75/0.5组成的脂质体在N/P比为10时，细胞活力下降了70%，而相同制剂在N/P比为2.5时是安全的。有趣的是，我们观察到在mRNA敲低效率方面存在差异，而每种制剂的转染率相当相似。含有50%DOPE的脂质体诱导的mRNA沉默约为80%。这项研究使我们能够突出关键参数，以开发安全且能诱导siRNA有效胞质释放的脂质复合物。
