Octaarginine- and Octalysine-modified Nanoparticles Have Different Modes of Endosomal Escape

Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-8012, Japan.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2008; 283(34):23450-61. DOI: 10.1074/jbc.M709387200
Source: PubMed


The present study examines the role of surface modification with an octaarginine peptide (R8) in liposomal escape from endocytic vesicles, using octalysine (K8) as a control cationic peptide; the mechanism of endosomal escape of liposomes was also investigated. Gene expression of condensed plasmid DNA encapsulated in R8-modified nanoparticles was more than 1 order of magnitude higher than that of K8-modified nanoparticles, and 2 orders of magnitude higher than gene expression using unmodified nanoparticles. The difference in gene expression could not be attributed to differences in uptake, as R8- and K8-modified liposomes were taken up primarily via macropinocytosis with comparable efficiency. The extent of R8-nanoparticle escape to the cytosol was double that of K8-nanoparticles. Suppression of endosome acidification inhibited R8-nanoparticle endosomal escape, but enhanced that of K8-nanoparticles. Using spectral imaging in live cells, we showed that R8- and K8-liposomes escaped from endocytic vesicles via fusion between the liposomes and the endosomal membrane. R8-liposomes fused efficiently at both acidic and neutral pH, whereas K8-liposomes fused only at neutral pH. Similar behavior was observed during in vitro lipid mixing and calcein-release experiments. Co-incubation of cells with distinctly labeled K8- and R8-modified nanoparticles confirmed a common uptake pathway and different rates of endosomal escape particularly at longer time intervals. Therefore, it was concluded that R8 on the liposome surface stimulates efficient escape from endocytic vesicles via a fusion mechanism that works at both neutral and acidic pH; in contrast, K8 mediates escape mainly at neutral pH.

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    • "Förster resonance energy transfer (FRET) has been exploited as a tool to monitor dissociation of DNA from the cationic liposome, because of fusion with anionic liposomes (Zelphati and Szoka, 1996). Through the use two labeled lipids within the cationic liposome acting as FRET donor and acceptor, FRET has also been used to monitor the fusion of liposomes with endocytic membranes, which leads to FRET cancelation (Wang and MacDonald, 2004; El-Sayed et al., 2008, 2009; Akita et al., 2009). However, these studies probe only the fusion of lipoplex and endosome membrane, failing to distinguish between the two crucial events required for cytosol access of DNA (DNA/lipid dissociation and DNA escape from the endosome compartment). "
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    ABSTRACT: Nonviral vectors are highly attractive for gene therapy from a clinical point of view, and cationic lipid nanoparticles in particular have generated considerable interest. However, despite considerable recent advances, problems associated with low transfection efficiencies remain to be resolved to fully meet the potential of these vectors. The trafficking of plasmid DNA from the extracellular space up to the nucleus is prevented by several barriers, including liposome/pDNA dissociation within the endosome and pDNA escape into the cytosol. The aim of this work was to develop and optimize a tool that could offer simultaneous quantitative information on both the intracellular dissociation of oligonucleotides from lipid nanoparticles, and on the DNA escape from endocytic compartments. The ability to follow in real time both of these processes simultaneously (in a quantitative manner), is expected to be of high value in the rationalization and conception of new lipid nanoparticle vectors for gene delivery for therapeutic purposes. To this effect, a combination of Förster Resonance Energy Transfer (FRET) and colocalization microscopy was employed. We show that it is possible to distinguish between liposome/pDNA dissociation and depletion of DNA within endosomes, providing resolution for the detection of intermediate species between endocytic particles with intact lipoplexes and endosomes devoid of DNA due to DNA escape or degradation. We demonstrate that after endocytosis, exceptionally few endocytic particles are found to exhibit simultaneously DNA/lipid colocalization and low FRET (DNA/Lipid dissociation). These results clearly point to an extremely short lived state for free plasmid within endosomes, which either escapes at once to the cytosol or is degraded within the endocytic compartment (due to exposure of DNA). It is possible that this limitation greatly contributes to reduction in probability of successful gene delivery through cationic lipid particles.
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    • "was used to determine whether fusion occurs between R8-modified liposomes (R8-Lip) and endocytic vesicles, with and without str-INF7 modification. The technique was followed exactly as described in a previous report [32]. Briefly, R8-Lip were prepared with an additional 1 mol% NBD-DOPE (excitation wavelength , 460 nm; emission wavelength, 534 nm) and 0.5 mol% rhodamine-DOPE (excitation wavelength, 550 nm; emission wavelength , 590 nm) as a donor and acceptor of fluorescence resonance energy transfer (FRET), respectively. "
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