Efficient Preparation of Giant Vesicles as Biomimetic Compartment Systems with High Entrapment Yields for Biomacromolecules

Authors

  • Takashi Kuroiwa,

    1. Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan (phone: +81-29-853-4627; fax: +81-29-853-4605)
    2. National Food Research Institute, National Agriculture and Food Research Organization, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan
    3. Faculty of Engineering, Tokyo City University, Tamazutsumi 1-28-1, Setagaya, Tokyo 158-8557, Japan
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  • Ryoji Fujita,

    1. Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan (phone: +81-29-853-4627; fax: +81-29-853-4605)
    2. National Food Research Institute, National Agriculture and Food Research Organization, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan
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  • Isao Kobayashi,

    1. National Food Research Institute, National Agriculture and Food Research Organization, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan
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  • Kunihiko Uemura,

    1. National Food Research Institute, National Agriculture and Food Research Organization, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan
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  • Mitsutoshi Nakajima,

    1. Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan (phone: +81-29-853-4627; fax: +81-29-853-4605)
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  • Seigo Sato,

    1. Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan (phone: +81-29-853-4627; fax: +81-29-853-4605)
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  • Peter Walde,

    Corresponding author
    1. Department of Materials, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, (phone: +41-44-6320473; fax: +41-44-6321265)
    • Department of Materials, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, (phone: +41-44-6320473; fax: +41-44-6321265)
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  • Sosaku Ichikawa

    Corresponding author
    1. Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan (phone: +81-29-853-4627; fax: +81-29-853-4605)
    • Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan (phone: +81-29-853-4627; fax: +81-29-853-4605)
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Abstract

The ‘lipid-coated ice-droplet hydration method’ was applied for the preparation of milliliter volumes of a suspension of giant phospholipid vesicles containing in the inner aqueous vesicle pool in high yield either calcein, α-chymotrypsin, fluorescently labeled bovine serum albumin or dextran (FITC-BSA and FITC-dextran; FITC=fluorescein isothiocyanate). The vesicles had an average diameter of ca. 7–11 μm and contained 20–50% of the desired molecules to be entrapped, the entrapment yield being dependent on the chemical structure of the entrapped molecules and on the details of the vesicle-formation procedure. The ‘lipid-coated ice droplet hydration method’ is a multistep process, based on i) the initial formation of a monodisperse water-in-oil emulsion by microchannel emulsification, followed by ii) emulsion droplet freezing, and iii) surfactant and oil removal, and replacement with bilayer-forming lipids and an aqueous solution. If one aims at applying the method for the entrapment of enzymes, retention of catalytic activity is important to consider. With α-chymotrypsin as first model enzyme to be used with the method, it was shown that high retention of enzymatic activity is possible, and that the entrapped enzyme molecules were able to catalyze the hydrolysis of a membrane-permeable substrate which was added to the vesicles after their formation. Furthermore, one of the critical steps of the method that leads to significant release of the molecules from the water droplets was investigated and optimized by using calcein as fluorescent probe.

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