Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, U.K.
Mol Pharm. 2012 Nov 5;9(11):3384-95. doi: 10.1021/mp300435x. Epub 2012 Oct 25.
Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N(4),N(9)-diacyl spermine conjugate, N(4)-linoleoyl-N(9)-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios of the neutral lipids, are reported. The effects of varying the lipid formulation and changing the N/P charge ratio on the intracellular delivery of siRNA to HeLa cells and on the siRNA-mediated gene silencing of a stably expressed reporter gene (EGFP) were evaluated. The presence of either cholesterol or DOPE in the mixture resulted in a marked increase in the delivery of the siRNA as well as enhanced EGFP silencing as evaluated by FACS. A LinOS/Chol 1:2 mixture resulted in the highest siRNA delivery and the most efficient EGFP silencing (reduced to 20%) at N/P = 3.0. Lowering the amount of siRNA from 15 pmol to 3.75 pmol, thus increasing the N/P charge ratio to 11.9, resulted in decreasing the amount of delivered siRNA, while the efficiency of gene silencing was comparable to that obtained with 15 pmol (N/P = 3.0) of siRNA. Mixtures of symmetrical N(4),N(9)-dioleoyl spermine (DOS) with cholesterol at 1:2 molar ratio showed less siRNA delivery than with LinOS/Chol at N/P = 3.0 (15 pmol of siRNA), and comparable delivery at N/P = 11.9 (3.75 pmol of siRNA). The EGFP silencing was comparable with LinOS and with DOS when mixed with cholesterol 1:2 (lipoplexes prepared with 15 pmol of siRNA), but LinOS mixtures showed better EGFP silencing when the siRNA was reduced to 3.75 pmol. Lipoplex particle size determination by DLS of cholesterol mixtures was 106-118 nm, compared to 194-356 nm for lipoplexes prepared with the spermine conjugates only, and to 685 nm for the LinOS/DOPE 1:1 mixture. Confocal microscopy showed successful siRNA delivery of red tagged siRNA and quantitative EGFP knockdown in HeLa EGFP cells; Z-stack photomicrographs showed that the delivered siRNA is distributed intracellularly. Cryo-TEM of siRNA LinOS/Chol 1:2 lipoplexes shows the formation of multilamellar spheres with a size of ∼100 nm, in good agreement with the particle size measured by DLS. The constant distance between lamellar repeats is ∼6 nm, with the electron-dense layers fitting a monolayer of siRNA. AlamarBlue cell viability assay showed that the lipoplexes resulted in cell viability ≥81%, with LinOS/Chol 1:2 mixtures resulting in cell viabilities of 89% and 94% at siRNA 15 nM and 3.75 nM respectively. These results show that lipoplexes of siRNA and LinOS/Chol mixtures prepared by the direct mixing of the lipid mixture and siRNA, without any preceding preformulation steps, result in enhanced siRNA delivery and EGFP knockdown, with excellent cell viability. Thus, LinOS/Chol 1:2 mixture is a promising candidate as a nontoxic nonviral siRNA vector.
本文报道了通过直接混合 siRNA 和非对称 N(4),N(9)-二酰基 spermine 缀合物 N(4)-亚油酰基-N(9)-油酰基-1,12-二氨基-4,9-二氮杂十二烷（LinOS）与胆固醇或 DOPE 的混合物来制备非病毒 siRNA 载体，其中中性脂质的摩尔比不同。研究了改变脂质配方和改变 N/P 电荷比对 HeLa 细胞中 siRNA 的细胞内传递以及稳定表达报告基因（EGFP）的 siRNA 介导基因沉默的影响。混合物中存在胆固醇或 DOPE 会导致 siRNA 的传递明显增加，并通过 FACS 评估增强 EGFP 沉默。LinOS/Chol 1:2 混合物在 N/P = 3.0 时导致最高的 siRNA 传递和最有效的 EGFP 沉默（降低至 20%）。从 15 pmol 降低至 3.75 pmol 的 siRNA 量，从而将 N/P 电荷比增加至 11.9，导致传递的 siRNA 量减少，而基因沉默的效率与用 15 pmol（N/P = 3.0）的 siRNA 获得的效率相当。具有 1:2 摩尔比的胆固醇的对称 N(4),N(9)-二油酰基 spermine（DOS）混合物在 N/P = 3.0 时显示的 siRNA 传递量低于 LinOS/Chol（15 pmol 的 siRNA），而在 N/P = 11.9 时的传递量则相当（3.75 pmol 的 siRNA）。当与胆固醇 1:2 混合时，EGFP 沉默与 LinOS 和 DOS 相当（用 15 pmol 的 siRNA 制备的脂质体），但当 siRNA 减少至 3.75 pmol 时，LinOS 混合物显示出更好的 EGFP 沉默。通过 DLS 测定胆固醇混合物的脂质体颗粒大小为 106-118nm，而仅用 spermine 缀合物制备的脂质体为 194-356nm，LinOS/DOPE 1:1 混合物为 685nm。共聚焦显微镜显示红色标记的 siRNA 的成功传递和 HeLa EGFP 细胞中定量 EGFP 敲低；Z 堆叠显微照片显示，递送至细胞内的 siRNA 是分布的。siRNA LinOS/Chol 1:2 脂质体的冷冻 TEM 显示形成具有约 100nm 大小的多层球体，与 DLS 测量的粒径非常吻合。层状重复之间的恒定距离约为 6nm，电子致密层适合单层 siRNA。AlamarBlue 细胞活力测定表明脂质体导致细胞活力≥81%，LinOS/Chol 1:2 混合物在 siRNA 15 nM 和 3.75 nM 时分别导致细胞活力为 89%和 94%。这些结果表明，通过直接混合脂质混合物和 siRNA 制备的 siRNA 和 LinOS/Chol 混合物的脂质体，无需任何预先的制剂步骤，可提高 siRNA 的传递和 EGFP 敲低，同时保持良好的细胞活力。因此，LinOS/Chol 1:2 混合物是一种有前途的非毒性非病毒 siRNA 载体候选物。
