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.

11 Reads
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Human Gene Therapy Methods 10/2014; 25(5). DOI:10.1089/hgtb.2014.080 · 2.44 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: An octaarginine-modified multifunctional envelope-type nano device (R8-MEND) was previously reported to be an efficient nonviral vector for the delivery of plasmid DNA, in vitro and after topical administration. We report herein on a novel stearylated derivative of the INF7 peptide, a derivative of the N-terminal domain of the HA2 protein of the influenza virus envelope, which enhances the endosomal escape of R8-MEND through a mechanism independent of fusion between the MEND coat and the endosomal membrane. The use of the novel peptide derivative would permit the gene expression of the R8-MEND to be improved, both in vitro and in vivo. R8-MEND modified with stearylated INF7 resulted in gene expression levels that were 77-fold higher than unmodified and 20-fold higher than the free INF7 peptide-modified R8-MEND with no cellular toxicity. Spectral imaging in live cells confirmed that the stearylated INF7 modification did not mediate fusion between liposomes and the endosomal membrane. The inclusion of DOPE to the R8-MEND coat was synergistic with the peptide in improving gene transfection. The intravenous injection of an R8-MEND modified with stearylated INF7 to ICR mice resulted in luciferase expression levels 240-fold higher in liver and 115-fold higher in spleen than that of the R8-MEND.
    Journal of Controlled Release 06/2009; 138(2):160-7. DOI:10.1016/j.jconrel.2009.05.018 · 7.71 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mitochondrial gene therapy has the potential for curing a variety of diseases that are associated with mitochondrial DNA mutations and/or defects. To achieve this, it will be necessary to deliver therapeutic agents into the mitochondria in diseased cells. A number of mitochondrial drug delivery systems have been reported to date. However, reports of mitochondrial-targeted DNA delivery are limited. To achieve this, the therapeutic agent must be taken up by the cell (1), after which, the multi-processes associated with intracellular trafficking must be sophisticatedly regulated so as to release the agent from the endosome and deliver it to the cytosol (2) and to pass through the mitochondrial membrane (3). We report herein on the mitochondrial delivery of oligo DNA as a model therapeutic using a Dual Function (DF)-MITO-Porter, an innovative nano carrier designed for mitochondrial delivery. The critical structural elements of the DF-MITO-Porter include mitochondria-fusogenic inner envelopes and endosome-fusogenic outer envelopes, modified with octaarginine which greatly assists in cellular uptake. Inside the cell, the carrier passes through the endosomal and mitochondrial membranes via step-wise membrane fusion. When the oligo DNA was packaged in the DF-MITO-Porter, cellular uptake efficiency was strongly enhanced. Intracellular observation using confocal laser scanning microscopy showed that the DF-MITO-Porter was effectively released from endosomes. Moreover, the findings confirmed that the mitochondrial targeting activity of the DF-MITO-Porter was significantly higher than that of a carrier without outer endosome-fusogenic envelopes. These results support the conclusion that mitochondrial-targeted DNA delivery using a DF-MITO-Porter can be achieved when intracellular trafficking is optimally regulated.
    Journal of Nanoparticle Research 08/2012; 14(8). DOI:10.1007/s11051-012-1013-3 · 2.18 Impact Factor
Show more


11 Reads
Available from