Characterisation of LMD virus-like nanoparticles self-assembled from cationic liposomes, adenovirus core peptide μ (mu) and plasmid DNA
Characterisation of LMD virus-like nanoparticles self-assembled from cationic liposomes, adenovirus core peptide μ (mu) and plasmid DNA
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DOI：
10.1038/sj.gt.3301686
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发表时间：
2002-05-01
期刊：
影响因子：
5.1
通讯作者：
Miller, AD
中科院分区：
文献类型：
--
作者：
Tagawa, T;Manvell, M;Miller, AD
脂质体：mu：DNA（LMD）是围绕与腺病毒的浓缩核心复合物相关的mu（mu）肽构建的三元核酸递送系统。LMD通过以1：0.6（w/w）的比例预缩合质粒DNA（D）与μ肽（M），然后将这些μ：DNA（MD）复合物与挤出的阳离子脂质体（L）组合，产生最终脂质来制备。mu：DNA比率为12：0.6：1（w/w/w）。正确的缓冲液条件、试剂浓度和混合速率对成功至关重要。然而，一旦建立了最佳条件，均匀的LMD颗粒（120+/-30 nm）将导致每个似乎包含包封在阳离子双层脂质体内的MD颗粒。LMD颗粒可以可重复地配制，它们适合于在-80 ℃下长期储存（>1个月），并且在质粒DNA浓度高达5 mg/ml（15 mM核苷酸浓度）时对聚集稳定。此外，在体外，LMD转染比阳离子脂质体-质粒DNA（LD）转染显著更有时间和剂量效率。短至10分钟的转染时间和低至0.001 μ g/孔的质粒DNA剂量导致显著的基因表达。LMD转染也将在存在生物流体（例如高达100%血清）的情况下进行，产生在不存在血清的情况下观察到的15-25%的基因表达水平。使用荧光标记的LMD颗粒的共聚焦显微镜实验的结果表明，内吞作用不是LMD转染的重要障碍，尽管核膜仍然是。我们还证实，通过LMD进行的体内局部肺转染在绝对值上至少等于通过GL-67：DOPE：DMPE-PEG（5）000（1：2：0.05 m/m/m）介导的转染，GL-67：DOPE：DMPE-PEG（5）000是一种公认的用于局部肺转染的“金标准”非病毒载体系统，并且实际上是至少六倍的剂量效率。所有这些特征使LMD成为一种重要的新的非病毒载体平台系统，通过系统的模块化升级过程，从该平台系统获得定制的非病毒递送系统。
Liposome:mu:DNA (LMD) is a ternary nucleic acid delivery system built around the mu (mu) peptide associated with the condensed core complex of the adenovirus. LMD is prepared by precondensing plasmid DNA (D) with mu peptide (M) in a 1:0.6 (w/w) ratio and then combining these mu:DNA (MD) complexes with extruded cationic liposomes (L) resulting in a final lipid.-mu:DNA ratio of 12:0.6:1 (w/w/w). Correct buffer conditions, reagent concentrations and rates of mixing are all crucial to success. However, once optimal conditions are established, homogeneous LMD particles (120+/-30 nm) will result that each appear to comprise an MD particle encapsulated within a cationic bilammellar liposome. LMD particles can be formulated reproducibly, they are amenable to long-term storage (>1 month) at -80degreesC and are stable to aggregation at a plasmid DNA concentration up to 5 mg/ml (15 mM nucleotide concentration). Furthermore, LMD transfections are significantly more time and dose efficient, in vitro than cationic liposome-plasmid DNA (LD) transfections. Transfection times as short as 10 min and plasmid DNA doses as low as 0.001 mug/well result in significant gene expression. LMD transfections will also take place in the presence of biological fluids (eg up to 100% serum) giving 15-25% the level of gene expression observed in the absence of serum. Results from confocal microscopy experiments using fluorescent-labelled LMD particles suggest that endocytosis is not a significant barrier to LMD transfection, although the nuclear membrane still is. We also confirm that topical lung transfection in vivo by LMD is at least equal in absolute terms with transfection mediated by GL-67:DOPE:DMPE-PEG(5)000 (1:2:0.05 m/m/m), an accepted 'gold-standard' non-viral vector system for topical lung transfection, and is in fact at least six-fold more dose efficient. All these features make LMD an important new non-viral vector platform system from which to derive tailor-made non-viral delivery systems by a process of systematic modular upgrading.