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

Abstract

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.

Cell penetrating peptide-mediated systemic siRNA delivery to the liver

“…(ii) the system delivers siRNA to the cytosol quite efficiently. Imaginig data revealed that most of the siRNAs were not colocalized in the lysosomal compartment (Fig.3), which is consistent with previous reports indicating that a high density of R8 peptide on the surface of nanoparticle facilitates the induction of macropinocytosis, which is an advantageous route for efficient intracellular trafficking, and which stimulates efficient endosomal escape (Khalil IA et al, 2006;El-Sayed A et al, 2008). (iii) there is no need to use chemically modified siRNA (Braasch DA et al, 2003) to protect against degradation in the serum because siRNA is efficiently encapsulated within a nanoparticle (Nakamura Y et al, 2007).…”

Octaarginine-modified liposomes enhance the anti-oxidant effect of Lecithinized superoxide dismutase by increasing its cellular uptake

“…It is assumed that endosomal escape would not be sufficient for achieving effective PC-SOD delivery. We recently reported that a liposome surface modification with the pH-sensitive fusogenic peptide, GALA, enhanced endosomal escape and permitted cytosolic delivery to be efficiently achieved [25,26,27]. Accordingly, it would be expected that the modification of GALA with R8-LP (PC-SOD) would show stronger anti-oxidant effects.…”

“…It has been reported that high density R8-LP is taken up efficiently by cells via macropinocytosis [21]. Moreover, R8-LP, composed of DOPE and PA, can achieve efficient endosomal escape, thus enhancing cytoplasmic delivery [25]. We first checked the effect of PC-SOD on the liposome preparation (Fig S1 in Supplementary material).…”

“…We attempted to construct R8-LP (PC-SOD) containing an endosome-fusogenic lipid composition (DOPE/PA = 7:2 (molar ratio)) [25]. It has been reported that high density R8-LP is taken up efficiently by cells via macropinocytosis [21].…”

Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter

“…Particle diameters were around 150 nm and the ζ-potentials were around + 30 mV. The outer envelope of all carriers had a endosome-fusogenic composition [27]. For the DF-MITO-Porter, the inner envelope had a mitochondria-fusogenic composition [19], while a non-mitochondrial fusogenic composition [19] was used in the control inner envelope.…”