Penetration of biomolecules into cells is of great importance in biotechnology such as in genetic modification, bio-imaging, delivery system etc. A numerous strategy has been used for effective penetration of nano-sized particles such as electroporation, sonication. However, these methods show highly cell damage. In order to develop low toxic and efficient process, we developed a new method called “freeze concentration” process. Freeze concentration is described as a physical phenomenon wherein at extreme low temperature; the water is transformed into ice-crystals leads to eject the solute molecules, which increases its concentration. We previously showed the effective use of freeze concentration process in effective protein delivery system.<sup>1), 2)</sup> Among the nanosize particles, liposomes are one of the most popular particles in biomedical application. However, liposomes exhibited instability that can limit its use. Therefore, conjugation of polymers in liposomes is highly attracted for retaining its stability for delivery system. The characterization of liposome was determined by DLS analysis. The sizes were in the range of 200-300 nm in diameter. The protein encapsulating efficiency in liposomes was determined by Bradford assay, the encapsulation efficiency of protein in polyampholyte-modified liposome was greater rather than bare liposome. An effective adsorption onto the cell membrane and internalization into cytosol of protein/liposome complex by facile freeze thawing method were confirmed by comparing with unfrozen cells (Fig.1). A new polyampholyte modified liposome was developed which has high efficacy in facilitating the endosomal escape to the cytoplasm of the cells with low toxicity. This evidences strongly prove that freeze concentration based strategy can be widely used not in cancer immunotherapy but also in gene therapy.

Intracellular delivery of biomolecules via freeze concentration using polyampholyte nanocarriers

2019 Spring National Meeting

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