Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures
Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures
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DOI：
10.1016/s0005-2736(00)00343-6
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发表时间：
2001-02-09
影响因子：
3.4
通讯作者：
Scherrer, P
中科院分区：
文献类型：
--
作者：
Semple, SC;Klimuk, SK;Scherrer, P
用于将反义寡脱氧核苷酸（ODN）和质粒DNA包封在脂质囊泡中的典型方法导致非常低的包封效率或采用当静脉内施用时表现出不利的药代动力学和毒性特征的阳离子脂质。在这项研究中，我们描述和表征了一种新的制剂工艺，该工艺利用可电离的氨基脂质（1，2-二油酰基-3-二甲基铵丙烷，DODAP）和含乙醇的缓冲系统，用于在脂质囊泡中包封大量（0.15-0.25 g ODN/g脂质）聚阴离子ODN。该方法需要存在高达40%的乙醇（v/v），并且初始制剂处于DODAP带正电荷的酸性pH值。此外，在配制过程中需要存在聚（乙二醇）-脂质以防止聚集。所形成的“稳定的反义脂质颗粒”（SALP）在将外部pH值调节至中性pH值时是稳定的，并且配制过程允许高达70%的包封效率。通过核酸酶保护测定和P-31 NMR测量确认ODN包封。冷冻电子显微镜表明，最终的颗粒组成的混合人口的单层和小的多层囊泡（80-140 nm直径），其相对比例是依赖于初始的ODN脂质比。最后，相对于游离反义ODN、阳离子脂质/ODN复合物和用季胺脂制备的SALP，SALP在小鼠中表现出显著增强的循环寿命。考虑到与其他制剂相比，该制剂的小颗粒尺寸和改善的包封效率、ODN与脂质的比率和循环时间，我们认为SALP代表了涉及核酸治疗剂的全身应用的可行候选物。(C)2001 Elsevier Science B. V.保留所有权利。
Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15-0.25 g ODN/g lipid) of polyanionic ODN in lipid vesicles. This process requires the presence of up to 40% ethanol (v/v) and initial formulation at acidic pH values where the DODAP is positively charged. In addition, the presence of a poly(ethylene glycol)-lipid was required during the formulation process to prevent aggregation. The 'stabilized antisense-lipid particles' (SALP) formed are stable on adjustment of the external pH to neutral pH values and the formulation process allows encapsulation efficiencies of up to 70%. ODN encapsulation was confirmed by nuclease protection assays and P-31 NMR measurements. Cryo-electron microscopy indicated that the final particles consisted of a mixed population of unilamellar and small multilamellar vesicles (80-140 nm diameter), the relative proportion of which was dependent on the initial ODN to lipid ratio. Finally, SALP exhibited significantly enhanced circulation lifetimes in mice relative to free antisense ODN, cationic lipid/ODN complexes and SALP prepared with quaternary aminolipids. Given the small particle sizes and improved encapsulation efficiency, ODN to lipid ratios, and circulation times of this formulation compared to others, we believe SALP represent a viable candidate for systemic applications involving nucleic acid therapeutics. (C) 2001 Elsevier Science B.V. All rights reserved.