Enhancement in selective mitochondrial association by direct modification of a mitochondrial targeting signal peptide on a liposomal based nanocarrier
Introduction
The efficient and site-specific delivery of therapeutic drugs is a critical issue in clinical nanotechnology. Receptor-targeted nanocarriers aimed at specific tissues and cells have been investigated by many researchers, and the findings have accelerated the application of nanotechnology in medicine (referred to as nanomedicine) (Juliano et al., 2009, Mohanty et al., 2011). However, these techniques have been limited to developing nanocarriers that target a specific organelle, such as the nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, etc., which are promising targets for therapeutic drugs. Mitochondria play a role in the homeostasis of vital physiological functions, including electron transfer, apoptosis and calcium storage (Szewczyk and Wojtczak, 2002). Mitochondrial dysfunction is implicated in a variety of human diseases, including mitochondrial inherited diseases, neurodegenerative disorders, diabetes mellitus and cancer (Chan, 2006, Reeve et al., 2008, Schapira, 2006, Wallace, 2005), therefore, mitochondria would be expected to be the target organelle for these diseases (Armstrong, 2007, Du and Yan, 2010, Fulda et al., 2010, Giorgi et al., 2012, Gogvadze et al., 2009, Yamada et al., 2007).
Effective medical therapies for such diseases will ultimately require an optimal mitochondrial delivery system, in which multiple processes i.e., cellular uptake, endosomal escape and mitochondrial targeting should be regulated. Many researches regarding cellular uptake and endosomal escape to improve the intracellular trafficking of the carrier have been performed, because these topics are common issues in the development of drug delivery system (Miyata et al., 2012, Pack et al., 2005, Wang et al., 2012, Yamada et al., 2012). On the other hand, the information about mitochondrial-targeted delivery of nano carrier has been limited, although many mitochondrial deliveries for various molecules have been reported (Mukhopadhyay and Weiner, 2007, Yamada and Harashima, 2008, Zhang et al., 2011), especially in the case of low molecular weight drugs (D'Souza et al., 2008, James et al., 2007, Wipf et al., 2005). The use of a mitochondrial targeting signal peptide (MTS) makes it possible to selectively deliver a variety of chemicals, proteins and linear DNA to mitochondria (Asayama et al., 2006, Flierl et al., 2003, Schatz, 1996). It is expected that MTS can be a useful ligand for selective mitochondrial delivery of nanoparticle. However, the direct modification of an MTS peptide to produce a nanoparticle for enhancing mitochondrial delivery has not been demonstrated.
The purpose of this study was to validate whether MTS can enhance the mitochondrial targeting of nanoparticles as well as endogenous mitochondrial proteins. We first synthesized a lipid derivative conjugated with MTS and then constructed MTS-modified liposomes. The mitochondrial binding activity of the prepared liposomes was then evaluated using isolated mitochondria. Mitochondrial targeting activity was also estimated using a cell homogenate, which is a better model of living cells than isolated mitochondria. Finally, we developed an innovative technology in which MTS and a MITO-Porter are integrated. The latter is a liposome-based nano carrier that delivers cargos to mitochondria via membrane fusion (Yamada and Harashima, 2008, Yamada et al., 2008, Yasuzaki et al., 2010) and verified the utility of the selectivity of MTS for mitochondrial delivery via membrane fusion.
Section snippets
Materials
Cholesterol (Chol), 1,2-Dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE), 7-nitrobenz-2-oxa-1, 3-diazole labeled DOPE (NBD-DOPE) and rhodamine-DOPE were purchased from Avanti Polar lipids (Alabaster, AL, USA). Egg yolk phosphatidyl choline (EPC) was obtained from Nippon Oil and Fats Co. (Tokyo, Japan). Sphingomyelin (SM) was purchased from Sigma (St. Louis, MO, USA). Stearyl octaarginine (STR-R8) was obtained from Kurabo Industries Ltd (Osaka, Japan). MTS peptide (NH2
Synthesis of MTS–DOPE
In this experiment, we used the MTS peptide derived from rat succinyl CoA synthetase (Henning et al., 1988) as a mitochondrial targeting ligand. We first confirmed that this MTS is functional in human cells, because we planned to investigate mitochondrial targeting activity using a cell homogenate prepared with human HeLa cells. The pTENG and pTENMG plasmids encoding the GFP and the MTS fused GFP were transfected into HeLa cells as described in the Supplementary Material. After transfection,
Discussion
In the previous study, we prepared R8-modified liposomes by just mixing of liposomes and stearyl R8 solutions (Yamada et al., 2008). We first attempted to prepare MTS-modified liposomes using the same procedure, however, the methods were not optimal in this case, because the MTS-DOPE was not soluble in water but in an organic solvent. Therefore, we prepared MTS-modified liposomes using lipid films containing MTS-DOPE formed by evaporation of the organic solvents in a glass tube, by the
Conclusion
Organelle targeting is one of the challenges in drug delivery. In this study, we reported an approach for mitochondrial targeting using surface modification of liposomes with an MTS peptide. Efficient accumulation of the MTS-modified liposomes on mitochondria was demonstrated using cell homogenate cocktails. In combination with mitochondrial fusion liposomes, MITO-Porter, the highly mitochondrial membrane fusion activity was indicated. These results would support our approach that a combination
Acknowledgment
This work was supported, in part by, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, Japan (NIBIO), a Grant-in-Aid for Young Scientists (A) and a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japanese Government (MEXT). We also thank Milton Feather for his helpful advice in writing the manuscript.
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