Cellular engineering via membrane fusogenic liposomes to functionalize membrane vesicles for biomedical applications

Abstract

Membrane vesicles (MVs) derived by many types of cells play an important role in cell to cell communication by transporting biological materials within the body, and they are also involved in various disease pathology. Especially in tumors, MVs are considered to be a contributing factor to immune evasion, angiogenesis, and metastasis of tumor. Therefore, engineering of MVs to load therapeutic compounds can be a potential tool to overcome the limitations of conventional nanotechnology and to cure diseases such as tumors. There have been many attempts to load therapeutic materials in MVs, but the damage of MVs, aggregation, and dissimilar characteristics between in vivo origin and in vitro origin MVs were limitations of those methods. Here, we suggest a novel method to engineer tumor cells instead of the MVs itself to secrete engineered MVs by using biocompatible membrane fusogenic liposomes (MFLs). MVs can be in situ engineered in the parent cells by synthetic liposomes carrying the therapeutic compounds and be secreted to translocate the compounds to neighboring cells. According to the results, hydrophilic and hydrophobic compounds delivered to the plasma membrane and cytosol of cells by MFLs were efficiently incorporated in the interior and membrane of MVs, respectively. Using this method, hydrophobic or hydrophilic therapeutic agents, functional molecules such as targeting peptides, and functional nanoparticles such as gold nanoparticles can be loaded or conjugated to MVs without any damage or change of characteristics. Furthermore, we applied this method to treat tumor cells. Importantly, we found that the hydrophobic photosensitizers delivered into the plasma membranes of peripheral cell layers of tumor spheroids in vitro and perivascular cell layers of tumor tissues in vivo respectively penetrated the spheroids and the tissues dramatically, thereby inducing significant phototherapeutic effects. We verified that such penetration of hydrophobic compounds was mediated by intercellular migration of MVs loaded with photosensitizers. Finally, we tested whether this method could be used in drug delivery to the retina. Model drugs loaded in MFLs dispersed well in the vitreous humor after intravitreal injection, penetrated whole layers of the retina by intercellular migration of MVs, and showed longer retention time than when the same compound was injected in the free form. This study demonstrates that cell-derived MVs, a natural transport system, can be engineered in the parent cells with synthetic liposomes to mediate intracellular migration of exogenous hydrophobic compounds over multiple cell layers both in vitro and in vivo. This MV-mediated delivery approach would significantly improve efficacy of therapeutic compounds in the poorly vascularized tumors and multilayered organs such as the retina. We believe that this work provides new insights to delivery and penetration of exogenous compounds in the tumor and retinal diseases.