Elsevier

Journal of Controlled Release

Volume 139, Issue 2, 15 October 2009, Pages 127-132
Journal of Controlled Release

A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo

https://doi.org/10.1016/j.jconrel.2009.06.008Get rights and content

Abstract

Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of both pDNA and siRNA. Modification of a MEND with polyuethylene glycol, i.e., PEGylation, is a potential strategy for in vivo delivery of MENDs to tumor tissue. However, PEGylation also inhibits both uptake and endosomal escape of MENDs. To overcome these limitations, we developed a PEG-peptide-DOPE (PPD) that can be cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, to further improve the silencing activity of encapsulated siRNA, we modified the PPD-MEND with a pH-sensitive fusogenic GALA peptide (GALA/PPD-MEND). First, we determined the GALA and PPD content that would optimize the synergistic functions of GALA and PPD. The most efficient gene silencing activity was achieved when GALA and either conventional PEG-lipid or PPD were used to modify the MEND at a molar ratio of 1:1. In this case, the silencing activity was comparable to that achieved when using a MEND that had not been modified with PEG (unmodified MEND). Furthermore, in vivo topical administration revealed that optimized PPD/GALA-MENDa resulted in more efficient gene silencing compared with unmodified MENDs. Collectively, data demonstrate that introduction of both of a pH-sensitive fusogenic GALA peptide and PPD into the MEND facilitates nanoparticle endosomal escape, thereby enhancing the efficiency of siRNA delivery and gene silencing.

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)

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