A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo
Graphical abstract
Introduction
RNA interference (RNAi), initiated by small interfering RNA (siRNA) is a promising strategy for the cure of human diseases [1], [2]. However, clinical trials of therapeutic siRNA are prohibited, because the efficiency of siRNA delivery into cytosol of target cells using a non-viral delivery system is inadequate [1], [2]. Efficient delivery of siRNA to the cytosol of target cells depends on both the translocation of the non-viral vectors through the plasma membrane and their subsequent escape from endosomal/lysosomal compartments [3], [4]. To overcome these barriers, a number of functional devices have been developed, including both ligands specific for cellular uptake and fusogenic peptides for enhancement of endosomal/lysosomal escape [5], [6]. In addition, many groups have developed various types of carrier systems for siRNA delivery, such as lipoplexes and polyplexes [7], [8], [9].
To control intracellular trafficking of the carrier and its cargo, we previously developed a multifunctional envelope-type nano device (MEND), in which nucleic acids are condensed using a polycation to form a core particle that is encapsulated in a lipid envelope [10], [11]. Modification of the envelope and/or MEND core with functional devices can be used to control the bio-distribution and intracellular trafficking of the MEND. For example, previously, we demonstrated that introduction of a pH-sensitive fusogenic GALA peptide (WEAALAEALAEALAEHLAEALAEALEALAA), i.e., cholesteryl-GALA, (Chol-GALA) [12] into MENDs, enhanced endosomal escape following internalization of MENDs via endocytosis, thereby enhancing the efficiency of gene transfer [13]. PEGylation of nanoparticles is known to enhance their half-life in systemic circulation [14]. Moreover, long-circulating nanoparticles, approximately 100–200 nm in diameter, accumulate efficiently in tumor tissue after systemic administration due to the enhanced permeability and retention (EPR) effect [15]. However, PEGylation has disadvantages: it inhibits both cellular uptake and subsequent endosomal escape of nanoparticles [16], [17]. Previously, we developed a PEG-peptide-DOPE ternary conjugate named PPD, which has a peptide sequence that is cleaved in the presence of matrix metalloproteinase in an attempt to overcome the limitations associated with PEG modification. The transfection activity of a MEND modified with PPD (PPD-MEND), which was dependent on matrix metalloproteinase cleavage of PEG, was greater than that of a MEND modified with non-cleavable PEG both in vitro and in vivo [18].
In the present study, we investigated the ability to co-modify a MEND with both PPD and GALA to control intracellular trafficking and consequently improve gene silencing both in vitro and in vivo. Our results suggest that manipulation of intracellular trafficking might allow successful delivery of siRNA both in vitro and in vivo.
Section snippets
Materials
Anti-luciferase siRNA (21-mer, 5′-GCGCUGCUGGUGCCAACCCTT-3′, 5′-GGGUUGGCACCAGCAGCAGCGCTT-3′) and anti-green fluorescent protein (GFP) siRNA (5′-GCUGACCCUGAAGUUCAUCTT-3′, GAUGAACUUCAGGGUCAGCTT-3′) were obtained from Thermo Electron GmbH (Ulm, Germany). Stearyl octaarginine (STR-R8) was synthesized as described previously [19]. Dioleoylphosphatidyl ethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), cholesterol, and distearoyl-sn-glycero-3-phoshoethanolamine-N-[methoxy
Characteristics of MENDs
The average diameter and ζ-potential of condensed siRNA particles were approximately 80 nm and − 20 mV, respectively. The average diameters and ζ-potentials of the prepared MENDs are summarized in Table 1. Unmodified MEND was 250 nm in diameter, and were positively charged due to the cationic lipid. The inversion of ζ-potential suggested that siRNA complex was encapsulated by the lipid envelope. PEG- and PPD-modification reduced the diameter of MENDs and the positive charge was decreased as
Discussion
These studies aimed to enhance the silencing activity of PEGylated MEND by encapsulating siRNA. The modification of PEG formed an aqueous layer on the surface of a MEND, which resulted in prolonging the systemic circulation of the MEND after intravenous administration due to escape from recognition and clearance by phagocytic cells of the reticuloendothelial system [14]. Therefore, PEGylation provides biocompatibility and a useful means for in vivo application. However, PEGylated MEND showed a
Conclusion
The results of the present study indicate that the combination of GALA or PPD synergistically improves the intracellular trafficking of non-viral vectors. In the in vitro study, the silencing efficiency of GALA/PPD-MENDs was comparable to that of unmodified MENDs and was significantly greater than that of PEG-MENDs. However, in vivo, GALA/PPD-MENDs exhibited greater gene silencing in tumor tissues compared with unmodified MEND. Collectively, co-modification of MENDs with both GALA and PPD
Acknowledgments
This work was financially supported by Grants-in-Aid for Scientific Research (A) and Grant-in-Aid for Young Scientists (Start-up) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Grants-in-Aid for Scientific Research on Priority Areas from the Japan Society for the Promotion of Science. We also thank Dr. J. L. McDonald for his helpful advice in editing the manuscript.
References (29)
- et al.
Pharmacokinetic/pharmacodynamic considerations in gene therapy
Drug Discov. Today
(2003) - et al.
A novel IRQ ligand-modified nano-carrier targeted to a unique pathway of caveolar endocytic pathway
J. Control. Release
(2008) - et al.
Efficient gene silencing in metastatic tumor by siRNA formulated in surface-modified nanoparticles
J. Control. Release
(2008) - et al.
Development of a non-viral multifunctional envelop-type nano device by a novel lipid film hydration method
J. Control. Release
(2004) - et al.
Multifunctional envelope-type nano device for non-viral gene delivery: concept and application of programmed packaging
J. Control. Release
(2007) - et al.
Tumor vascular permeability and the EPR effect in macromoleculr therapeutics: a review
J. Control. Release
(2000) - et al.
PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles
Eur. J. Cell Biol.
(2004) - et al.
Pegylation of liposomes favours the endosomal degradation of the delivered phosphodiester oligonucleotides
J. Control. Release
(2007) - et al.
Embodying a stable alpha-helical protein structure through efficient chemical ligation via thioether formation
Bioorg. Med. Chem.
(1997) - et al.
Octaarginine-modified multifunctional envelope-type nano device for siRNA
J. Control. Release
(2007)
Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo
J. Biol. Chem.
Roles of lipid polymorphism in intracellular delivery
Adv. Drug Deliv. Rev.
Acid-triggered release via dePEGylation of DOPE liposomes containing acid-labile vinyl ether PEG-lipids
J. Control. Release
Liposomes with detachable polymer coating: destabilization and fusion of dioleoylphosphatidylethanolamine vesicles triggered by cleavage of surface-grafted poly(ethylene glycol)
FEBS Lett.
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