Microvesicle-encapsulated oncolytic virus: preparation, identification, and killing effect on Hepa 1-6 cells in vitro

Abstract:

[Background] Oncolytic virus therapy is a new direction in the research on tumor treatment, and Newcastle disease virus (NDV) has attracted much attention because of its high biosafety and accurate targeting. However, viral clearance by neutralizing antibodies will weaken the sustained effect of oncolytic virus. The tumor cell-derived microvesicle delivery system is expected to break the above bottleneck. [Objective] To establish the tumor cell-derived microvesicle delivery system of oncolytic NDV and evaluate its antitumor effect in vitro. [Methods] Microvesicle-encapsulated NDV (MV@NDV) was extracted by differential centrifugation. The particle sizes of MV, NDV, and MV@NDV were measured by a particle size meter. The relative positions of MV and NDV in MV@NDV were observed by transmission electron microscopy. Western blotting (WB) was employed to determine the protein levels of Integrin β-1, Gpc 3, and Flotillin-1 of MV@NDV. Laser confocal microscopy was employed to observe MV@NDV 24 h post infection in Hepa 1-6 cells and the expression of NDV-HN protein in cells. An inverted microscope and the cell counting kit-8 (CCK-8) were used to examine the changes the morphology and viability of infected Hepa 1-6 cells 24, 48, and 72 h post infection, respectively. Quantitative real-time polymerase chain reaction (qPCR) and WB were employed to determine the mRNA levels of apoptosis-associated genes Caspase-3 and Caspase-9 and the protein level of Caspase-3, respectively, in the MV@NDV-infected Hepa 1-6 cells 24 h post infection. [Results] The particle sizes of MV, NDV, and MV@NDV were within the ranges of 141−342 nm, 91−825 nm, and 164−712 nm, respectively. WB results indicated that Integrin β-1, Gpc 3, and Flotillin-1 were expressed in MV@NDV. The Hepa 1-6 cells infected with MV@NDV and NDV showed red fluorescence and expressed NDV-HN protein. In addition, the viability of Hepa 1-6 cells decreased in the MV@NDV group compared with that in the NC group (P<0.05). The Hepa 1-6 cells in the MV@NDV group showed up-regulated mRNA levels of Caspase-3 and Caspase-9 and up-regulated protein level of Caspase-3 compared with the NC group (P<0.05). [Conclusion] The successful construction of NDV microvesicle delivery system MV@NDV is expected to solve the bottleneck of therapeutic oncolytic Newcastle disease virus (NDV) in the clinical translation, and provide new ideas and rationale for clinical NDV anti-tumor aspects.

Key words:oncolytic virus;Newcastle disease virus;microvesicles;anti-tumor