Rab11 and Lysotracker Markers Reveal Correlation between Endosomal Pathways and Transfection Efficiency of Surface-Functionalized Cationic Liposome-DNA Nanoparticless
Rab11 and Lysotracker Markers Reveal Correlation between Endosomal Pathways and Transfection Efficiency of Surface-Functionalized Cationic Liposome-DNA Nanoparticless
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DOI：
10.1021/acs.jpcb.6b04441
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发表时间：
2016-07-07
影响因子：
3.3
通讯作者：
Safinya, Cyrus R.
中科院分区：
文献类型：
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作者：
Majzoub, Ramsey N.;Wonder, Emily;Safinya, Cyrus R.
阳离子脂质体（CL）作为DNA和短干扰RNA的载体被广泛研究，用于基因递送和沉默，相关的临床试验正在进行中。转染效率（TE）的优化需要理解CL核酸纳米颗粒（NP）与细胞的相互作用、NP内体途径、内体逃逸和导致活性核酸从脂质载体释放的事件。在这里，我们研究了内体途径和TE的表面功能化的CL-DNA纳米粒子在PC-3前列腺癌细胞展示过表达的整合素和神经纤毛蛋白-1受体。NP含有RGD-PEG-脂质或RPARPAR-PEG-脂质，分别靶向整联蛋白和神经纤毛蛋白-1受体）或对照PEG脂质。使用Rab 11-GFP和Lysotracker的荧光共定位使得能够在低（10 mol %）、高（50 mol %）和非常高（70 mol %）膜电荷密度（sigma（M））下以增加的五价MVL 5（+5 e）摩尔分数同时共定位具有再循环内体（Rab 11）和晚期内体/溶酶体（Rab 7/Lysotracker）途径的NP。对于这些阳离子NP（脂质/DNA摩尔电荷比，rho（chg）= 5），膜电荷密度对途径选择和转染效率的影响对于两种肽PEG NP是相似的，尽管在定量上，与RPARPAR-PEG NP相比，RGD-PEG的影响更大。在低σ（M）下，肽PEG NP显示对再循环内体的偏好超过晚期内体/溶酶体途径。σ（M）从低到高的增加导致与再循环内体的共定位减少，并且与晚期内体/溶酶体途径的共定位同时增加。结合在低和高上午共定位和功能TE数据表明，较高的TE与更大比例的NP共定位与晚期内体/溶酶体途径，而较低的TE与更大比例的NP共定位与Rab 11再循环途径。这些发现导致一种假设，即σ（M）的增加，导致增强的晚期内体/溶酶体途径选择和更高的TE，是由于NP和内体腔膜之间的非特异性静电吸引力增加，相反，NP的增强的再循环途径和更低的TE是由于较弱的吸引力。令人惊讶的是，在非常高的sigma（M）下，在低和高sigma（M）下观察到的两个途径之间的反比关系破裂，指向更复杂的NP途径行为。
Cationic liposomes (CLs) are widely studied as carriers of DNA and short-interfering RNA for gene delivery and silencing, and related clinical trials are ongoing. Optimization of transfection efficiency (TE) requires understanding of CL nucleic acid nanoparticle (NP) interactions with cells, NP endosomal pathways, endosomal escape, and events leading to release of active nucleic acid from the lipid carrier. Here, we studied endosomal pathways and TE of surface-functionalized CL-DNA NPs in PC-3 prostate cancer cells displaying overexpressed integrin and neuropilin-1 receptors. The NPs contained RGD-PEG-lipid or RPARPAR-PEG-lipid, targeting integrin, and neuropilin-1 receptors, respectively) or control PEG lipid. Fluorescence colocalization using Rab11-GFP and Lysotracker enabled simultaneous colocalization of NPs with recycling endosome (Rab11) and late endosome/lysosome (Rab7/Lysotracker) pathways at increasing mole fractions of pentavalent MVL5 (+5 e) at low (10 mol %), high (50 mol %), and very high (70 mol %) membrane charge density (sigma(M)). For these cationic NPs (lipid/DNA molar charge ratio, rho(chg) = 5), the influence of membrane charge density on pathway selection and transfection efficiency is similar for both peptide PEG NPs, although, quantitatively, the effect is larger for RGD-PEG compared to RPARPAR-PEG NPs. At low sigma(M), peptide PEG NPs show preference for the recycling endosome over the late endosome/lysosome pathway. Increases in sigma(M), from low to high, lead to decreases in colocalization with recycling endosomes and simultaneous increases in colocalization with the late endosome/lysosome pathway. Combining colocalization and functional TE data at low and high am shows that higher TE correlates with a larger fraction of NPs colocalized with the late endosome/lysosome pathway while lower TE correlates with a larger fraction of NPs colocalized with the Rab11 recycling pathway. The findings lead to a hypothesis that increases in sigma(M), leading to enhanced late endosome/lysosome pathway selection and higher TE, result from increased nonspecific electrostatic attractions between NPs and endosome luminal membranes, and conversely, enhanced recycling pathway for NPs and lower TE are due to weaker attractions. Surprisingly, at very high sigma(M), the inverse relation between the two pathways observed at low and high sigma(M) breaks down, pointing to a more complex NP pathway behavior.