Introduction: To control the targeting specificity of liposomes, immunoliposomes, which are mainly prepared by chemically conjugation of antibody to lipid, were developed. While the high practical potential, there are still problems in the methods of the preparations. In previous studies, we developed liposome-chaperoning technology to obtain proteoliposomes by cell-free membrane protein synthesis[1],[2]. In this system, we found that expressing membrane protein was spontaneously integrated into liposomal membrane with functional form. We designed here single-chain transmmbrane antibody (scTab) against epidermal growth factor receptor (EGFR), and directly reconstituted scTab into liposomes by cell-free membrane protein synthesis. We also reported the functions of scTab-integrated liposome.
Materials and Methods: Plasmid DNA construction: pURE-antiEGFR scTab was constructed by inserting the polymerase chain reaction amplificon encoding antiEGFR scTab into the pURE1 vector. Preparation: Liposomes were prepared using natural swelling method. The cell-free synthesis of scTab was performed with or without the final concentration of 10 mM liposomes. The proteoliposomes were purified by density gradient ultracentrifugation. Incorporation efficiency was evaluated by performing the western blotting of each fraction after ultracentrifugation. The binding affinities and specificities to EGFR: Binding affinities of scTab/liposomes were measured by ELISA. In brief, scTab/liposomes were immobilized into 96well-plate, subsequently EGFR were applied into each well. Binding specificities were evaluated by using confocal laser scanning microscope and flow cytometer after incubation scTab/liposomes with EGFR expressing cell lines.
Results and Discussion: In the absence of liposomes, all synthesized scTab was aggregated. In contrast, in the presence of liposomes, scTab synthesized was detected in the liposome fraction (approx. 60 %). The result indicated that liposome effectively prevented the aggregation of scTab. Subsequently, we examined whether scTab/liposomes bound to EGFR or not by ELISA. The amount of EGFR bound to scTab/liposomes increased upon the increase of EGFR concentration. The results suggest that scTab synthesized was incorporated into liposome and folded to the correct conformation with the function of antibody. Finally, we evaluated cell specific affinities of scTab/liposomes by using cell lines with different expression levels of EGFR. The amount of cells bound to scTab/liposomes was dependent on EGFR expression levels of cell lines. The scTab/liposome acted as an immunoliposome.
Conclusion: We proposed new preparation method of immunoliposome with single chain Fv by using liposome/cell-free membrane protein synthesis systems (liposome-chaperoning technology).
This work was supported by the Exploratory Research for Advanced Technology Department of the Japan Science and Technology Agency (JST-ERATO)
References:
[1] M. Kaneda et al., Biomaterials, 30, 3971-3977 (2009).
[2] Y. Moritani et al., FEBS J., 277, 3343-3352 (2010).