Mechanism of Polyplex- and Lipoplex-Mediated Delivery of Nucleic Acids: Real-Time Visualization of Transient Membrane Destabilization without Endosomal Lysis

Abstract

Lipoplexes and polyplexes are widely applied as nonviral gene delivery carriers. Although their efficiencies of transfection are comparable, their mechanisms of delivery, specifically at the level of nucleic acid release from endosomes, are different. Thus, lipoplex-mediated release is proposed to rely on lipid mixing, as occurs between lipoplex and endosomal target membrane, the ensuing membrane destabilization leading to nucleic acid delivery into the cytosol. By contrast, the mechanism by which polyplexes, particularly those displaying a high proton buffering capacity, release their nucleic acid cargo from the endosome, is thought to rely on a so-called "proton sponge effect", in essence an osmotically induced rupturing of the endosomal membrane. However, although a wealth of indirect insight supports both these mechanisms, direct evidence is still lacking. Therefore, to further clarify these mechanisms, we have investigated the interaction of lipo- and polyplexes with HeLa cells by live cell imaging. As monitored over an incubation period of 2 h, our data reveal that in contrast to the involvement of numerous nanocarriers in case of lipoplex-mediated delivery, only a very limited number of polyplexes, that is, as few as one up to four/five nanocarriers per cell, with an average of one/two per cell, contribute to the release of nucleic acids from endosomes and their subsequent accumulation into the nucleus. Notably, in neither case complete rupture of endosomes nor release of intact polyplexes or lipoplexes into the cytosol was observed. Rather, at the time of endosomal escape both the polymer and its genetic payload are separately squirted into the cytoplasm, presumably via (a) local pore(s) within the endosomal membrane. Specifically, an almost instantaneous and complete discharge of nucleic acids and carrier (remnants) from the endosomes is observed. In case of lipoplexes, the data suggest the formation of multiple transient pores over time within the same endosomal membrane, via which the cargo is more gradually transferred into the cytosol.

Genome Editing for Cancer Therapy: Delivery of Cas9 Protein/sgRNA Plasmid via a Gold Nanocluster/Lipid Core–Shell Nanocarrier

“…On the one hand, DOTAP is a monovalent cationic lipid. Previous reports indicated that the ion‐pair effects led to efficient endosomal escape in lipid‐based formulations 26, 27. In our work, the ion‐pairs might form between the cationic DOTAP and the anionic lipids on the endosome membrane, which can induce the disassembly of LGCP and allow the release of the complex of TAT‐GNs/Cas9 protein/sgPlk1 plasmid from LGCP.…”

Fluorescence Imaging of siRNA Delivery by Peptide Nucleic Acid-based Probe

“…17,18,21,24 These kinetics and intracellular behaviors are in good agreement with the results using similar carriers. [23][24][25] In our previous study, Py-AA-TO was shown to selectively visualize the siRNA encapsulated in a polymer-based carrier. 11 We thus examined whether the fluorescence signal of Py-AA-TO could be a good indicator to assess not only cellular uptake but also the disassembly of the polyplex.…”

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

“…In that case, fluorescence will only return once the polynucleotides have dissociated from their carrier. This principle was elegantly explored to develop an endosomal escape assay based on the (de)quenching of small oligonucleotide fragments 168 . By simultaneously labeling different types of endosomal vesicles, the specific type of endosomes from which complexes are preferentially released has been recently identified 169 .…”