Elsevier

Methods in Enzymology

Volume 509, 2012, Pages 301-326
Methods in Enzymology

Chapter fifteen - Multifunctional Envelope-Type Nano Device (MEND) for Organelle Targeting Via a Stepwise Membrane Fusion Process

https://doi.org/10.1016/B978-0-12-391858-1.00015-0Get rights and content

Abstract

A single cell contains a variety of organelles. Included among these organelles are the nucleus that regulates the central dogma, mitochondria that function as an energy plant, the Golgi apparatus that determines the destination of endogenous protein, and others. If it were possible to prepare a nano craft that could specifically target a specific organelle, this would open a new field of research directed toward therapy for various diseases. We recently developed a new concept of “Programmed Packaging,” by which we succeeded in creating a multifunctional envelope-type nano device (MEND) as a nonviral gene-delivery system. Our attempts to target certain organelles (nucleus and mitochondria) are described here, mainly focusing on the construction of a tetra-lamellar MEND (T-MEND), and on methods for screening the organelle-specific fusogenic envelope. The critical structural elements of the T-MEND include an organelle-specific membrane-fusogenic inner envelope and a cellular membrane-fusogenic outer envelope. The resulting T-MEND can be utilized to overcome intracellular membrane barriers, since it involves stepwise membrane fusion. To deliver cargos into a target organelle in our strategy, the carriers must fuse with the organelle membrane. Therefore, we screened a series of lipid envelopes that have the potential for fusing with an organelle membrane by monitoring the inhibition of fluorescence resonance energy transfer and identified the optimal lipid conditions for nuclear and mitochondrial membrane fusion. Finally, we describe the delivery of a bioactive molecule targeted to the nucleus and mitochondria in living cells, demonstrating that this system can be useful for targeting various organelles.

Section snippets

Programmed Packaging Concept and Construction of R8-MEND

We recently developed a multifunctional envelope-type nano device (MEND) based on a new packaging concept called “Programmed Packaging” (Kogure et al., 2004, Kogure et al., 2008), in which various functional devices that control intracellular trafficking are packaged into single nanoparticles so as to permit them to function at the appropriate place and time. This concept consists of three components: (1) a program to overcome all barriers, (2) design of functional devices and their

Screening of Lipid Compositions for Their Ability to Fuse with Nuclear and Mitochondrial Membranes

Targeted delivery of an engineered gene or gene product to the nucleus or a mitochondrion is an essential first step toward the therapeutic restoration of a missing cellular function. The nuclear localization signal (NLS) peptide can be used to guide a protein to the nucleus (Yoneda et al., 1992). However, the same NLS does not function as such when attached to a pDNA (Nagasaki et al., 2003, Tanimoto et al., 2003): the positively charged NLS can be neutralized by the anionic pDNA, and it is

Construction of Tetra-Lamellar MEND (T-MEND)

As described above, the R8-MEND is capable of inducing macropinocytosis and can escape lysosomal degradation, leading to transfection activities as high as that for adenovirus in dividing cells (Khalil et al., 2007). However, in nondividing cells, the ultimate barrier, namely the nuclear membrane, must also be overcome. The R8-MEND cannot in corporate two kinds of envelopes with different compositions for different membrane fusions. To solve this problem, an innovative nanotechnology was

The nuclear membrane, an ultimate barrier to gene delivery to the nonmitotic cells

For a successful gene-delivery system, the nuclear membrane is the ultimate barrier that must be overcome. One of the typical results revealing its barrier function has been observed in the cell-to-cell variation in transgene expression. Flow cytometry analyses revealed that the percent of marker gene expression-positive cells to all cells analyzed was sharply enhanced when the cell cycle progressed through the M-phase (Tseng et al., 1999). Many investigators believe that the M-phase-specific

Mitochondrial Bioactive Molecule Delivery Using a Dual Function-MITO-Porter as a MEND for Mitochondrial Delivery

Mitochondrial dysfunction has recently been implicated in a variety of diseases (Chen and Chan, 2009, Kyriakouli et al., 2008, Reeve et al., 2008, Schapira, 2006, Tuppen et al., 2010, Wallace, 2005). Mutations and defects of mitochondrial DNA (mtDNA) are thought to be causes of mitochondrial diseases. Therefore, mitochondrial gene therapy would be expected to be useful and productive for the treatment of various diseases. To achieve such an innovative therapy, it will be necessary to deliver

Conclusions

In summary, we propose a novel strategy for the targeted delivery of macromolecules to a specific organelle (i.e., nucleus and mitochondria) based on the concept of stepwise membrane fusion, which is achieved using multicoated nanoparticles with endosome- and organelle-fusogenic lipid envelopes. At this time, a double-layered coating is added in a stepwise manner, based on the fusion of SUVs. Thus, in principle, only a particle with even numbers of lipid envelopes can be prepared. In the

Acknowledgments

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), Funding Program for Next Generation World-Leading Researchers (NEXT Program), 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). H.A. is also supported by the Asahi Glass Foundation. We

References (70)

  • K. Kogure et al.

    Development of a non-viral multifunctional envelope-type nano device by a novel lipid film hydration method

    J. Control. Release

    (2004)
  • K. Kogure et al.

    Multifunctional envelope-type nano device (MEND) as a non-viral gene delivery system

    Adv. Drug Deliv. Rev.

    (2008)
  • R.J. Lee et al.

    Folate-targeted, anionic liposome-entrapped polylysine-condensed DNA for tumor cell-specific gene transfer

    J. Biol. Chem.

    (1996)
  • W. Li et al.

    GALA: A designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery

    Adv. Drug Deliv. Rev.

    (2004)
  • O. Maier et al.

    Fluorescent lipid probes: Some properties and applications (a review)

    Chem. Phys. Lipids

    (2002)
  • T. Masuda et al.

    Evaluation of nuclear transfer and transcription of plasmid DNA condensed with protamine by microinjection: The use of a nuclear transfer score

    FEBS Lett.

    (2005)
  • A. Mesika et al.

    A regulated, NFkappaB-assisted import of plasmid DNA into mammalian cell nuclei

    Mol. Ther.

    (2001)
  • R. Moriguchi et al.

    A multifunctional envelope-type nano device for novel gene delivery of siRNA plasmids

    Int. J. Pharm.

    (2005)
  • Y. Nakamura et al.

    Octaarginine-modified multifunctional envelope-type nano device for siRNA

    J. Control. Release

    (2007)
  • F. Nicol et al.

    Effect of cholesterol and charge on pore formation in bilayer vesicles by a pH-sensitive peptide

    Biophys. J.

    (1996)
  • H. Pollard et al.

    Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells

    J. Biol. Chem.

    (1998)
  • A.H. Schapira

    Mitochondrial disease

    Lancet

    (2006)
  • S.M. Shaheen et al.

    KALA-modified multi-layered nanoparticles as gene carriers for MHC class-I mediated antigen presentation for a DNA vaccine

    Biomaterials

    (2011)
  • Y. Shinohara et al.

    Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: Dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation

    Biochim. Biophys. Acta

    (1997)
  • W.C. Tseng et al.

    Mitosis enhances transgene expression of plasmid delivered by cationic liposomes

    Biochim. Biophys. Acta

    (1999)
  • H.A. Tuppen et al.

    Mitochondrial DNA mutations and human disease

    Biochim. Biophys. Acta

    (2010)
  • D.C. Wallace

    The mitochondrial genome in human adaptive radiation and disease: On the road to therapeutics and performance enhancement

    Gene

    (2005)
  • N. Wiedemann et al.

    The protein import machinery of mitochondria

    J. Biol. Chem.

    (2004)
  • Y. Yamada et al.

    Mitochondrial drug delivery systems for macromolecule and their therapeutic application to mitochondrial diseases

    Adv. Drug Deliv. Rev.

    (2008)
  • Y. Yamada et al.

    MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion

    Biochim. Biophys. Acta

    (2008)
  • Y. Yamada et al.

    Dual function MITO-Porter, a nano carrier integrating both efficient cytoplasmic delivery and mitochondrial macromolecule delivery

    Mol. Ther.

    (2011)
  • Y. Yoneda et al.

    A long synthetic peptide containing a nuclear localization signal and its flanking sequences of SV40 T-antigen directs the transport of IgM into the nucleus efficiently

    Exp. Cell Res.

    (1992)
  • I. Ben-Efraim et al.

    Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

    J. Cell Biol.

    (2001)
  • G. Breuzard et al.

    Nuclear delivery of NFκB-assisted DNA/polymer complexes: Plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging

    Nucleic Acids Res.

    (2008)
  • S. Brunner et al.

    Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus

    Gene Ther.

    (2000)
  • Cited by (42)

    • Introduction to nanomedicine an overview

      2021, Nanomedicine Manufacturing and Applications
    View all citing articles on Scopus
    1

    These authors contributed equally.

    View full text