Microvesicles are a type of a cell organelle generally formed in the size of 0.03-1 um that are naturally released from a cell membrane in almost all cell types to be formed in a shape of a phospholipid bilayer and contain intracellular components such as messenger ribonucleic acid (mRNA), deoxyribonucleic acid (DNA), and protein.
As a basic tool of a cell for metabolism, transportation of a metabolic substance, storage of enzymes, chemical reactions, and the like, the microvesicles mediate intercellular signaling by delivering mRNA, microRNA (miRNA), and protein among cells. For example, microvesicles derived from a cancer cell induce changes in surrounding normal cells by delivering oncoprotein or miRNA. In addition, by being directly released from the cell membrane, the microvesicles hold an antigenic substance of a mother cell, thereby also being utilized in research for developing a vaccine. Besides, the microvesicles also play a role of removing misfolded protein, a cytotoxic substance or byproducts generated by intracellular metabolism.
Based on the intercellular signaling function of the microvesicles, a study has been actively carried out to develop a technology of delivering a drug or biomaterial to cells using an artificial phospholipid bilayer. The artificial microvesicles are widely utilized in biological experiments due to advantages of having high delivery efficiency and robustly protecting a substance to be delivered from an external environment.
However, artificial microvesicles prepared from the phospholipids purified from beans or eggs do not contain membrane protein, thus being unsuitable for inducing cell fusion. In addition, a process of removing the organic solvents is essential because surfactants and organic solvents used in preparing artificial microvesicls are toxic. Consequently, a method for preparing artificial microvesicles that are more serviceable in cell based experiments is required.
Recently, a technology of preparing artificial microvesicles by applying methods such as a conventional extrusion process or sonication process to a cell suspension to divide cells into small fragments and utilizing the artificial microvesicles in disease diagnosis or drug delivery has been disclosed (Title of Invention: Microvesicles derived from mammalian nucleated cell and use thereof, Inventor: GHO, Yong Song, Patent Application No. 10-2010-0063372). In addition, the inventors of the present invention have also developed a method of preparing artificial microvesicles from embryonic stem cells by a conventional extrusion method and treating somatic cells with the artificial microvesicles to induce the somatic cells to become induced pluripotent stem cells (Title of Invention: Method for preparing induced pluripotent stem cells using artificial microvesicles derived from embryonic stem cells, Inventor: PARK, Jae-Sung, Patent Application No. 10-2011-0040202).
In case of the preparing method mentioned above, the prepared artificial microvesicles mimic the structure of a cell membrane due to being derived from the cell membrane. However, a buffer solution etc. used during the preparation is included in the artificial microvesicles, causing cytoplasm itself to be diluted and a concentration of a target substance to be lowered as a result, thereby not only requiring a greater amount of artificial microvesicles to accurately diagnose a disease and deliver a predetermined amount of the target substance, but also having a problem of causing loss in an intracellular substance.
Accordingly, to solve the problems that may occur while preparing artificial microvesicles by conventional methods, the present inventors have developed an apparatus for preparing artificial microvesicles using centrifugal force, and completed the present invention by confirming that the artificial microvesicles prepared by the apparatus contain more intracellular components than the artificial microvesicles prepared by the conventional methods.