Rab11 and Lysotracker Markers Reveal Correlation between Endosomal Pathways and Transfection Efficiency of Surface-Functionalized Cationic Liposome–DNA Nanoparticles

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• 作者：Ramsey N. Majzoub ; Emily Wonder ; Kai K. Ewert ; Venkata Ramana Kotamraju ; Tambet Teesalu ; Cyrus R. Safinya
• 刊名：Journal of Physical Chemistry B
• 出版年：2016
• 出版时间：July 7, 2016
• 年：2016
• 卷：120
• 期：26
• 页码：6439-6453
• 全文大小：968K
• 年卷期：0
• ISSN：1520-5207

文摘

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 (σ_M). For these cationic NPs (lipid/DNA molar charge ratio, ρ_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 σ_M, peptide–PEG NPs show preference for the recycling endosome over the late endosome/lysosome pathway. Increases in σ_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 σ_M 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 σ_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 σ_M, the inverse relation between the two pathways observed at low and high σ_M breaks down, pointing to a more complex NP pathway behavior.