Alginate-Based Microencapsulation Of Human Embryonic Kidney 293 Cells To Release Lefty For Tumor Reversal Treatment

Recent study showed that tumor cells can be reversed to phenotype normal by epigenetically mechanism. Lefty protein, the inhibitor of nodal, played critical role during the process. The lefty exhibited high level expression in embryonic tissue while showed low level expression in normal adult tissue and malignant tumor such as breast cancer and melanoma. Current recombinant lefty protein made by prokaryotic expression system showed much lower biological effect due to lack of glycosylation modification. Eukaryotic expression system confronted with low production, high price, and inconvenient clinical application. Therefore, to solve the application of lefty for tumor treatment is the key question. In this study, we propose the strategy of local controlled release of lefty protein by encapsulation of expression lefty cells. In this study,pCDNA3.1-LEFTY1 and pIRES-GFP-LEFTY2 were constructed firstly, and by cell transfection technique two HEK 293 cell line that can stably express recombinant Lefty 1 and 2 was established respectively. The lefty-1 and lefty-2 protein can be detected by western blot in the cell culture medium which demonstrated that the protein can be secreted into the medium.Previous study showed the lack of cell adhesion molecular on the backbone of alginate polymer. In this study, gelatin was used to modify alginate and a complex capsule made by alginate and gelatin was obtained and the cell viability of HEK 293 was improved. Furthermore, an apparatus of electrostatic microencapsulation was fabricated and with this equipment, the capsule with 100μm diameter can be successfully fabricated.The lefty release system was established by alginate-based microencapsulation of lefty stably secreting 293 cell line. The lefty release system was implanted in the CD1 mouse for monitor the lefty release in vivo. ELISA technique was used to detect the concentration of lefty-1 protein in the mouse serum. By co-culture of lefty release capsule with breast cancer cell line MDA-MB231, the effect of released lefty on the MDA-MB231 was preliminarily studied. The results showed that released lefty protein can inhibit the proliferation of breast cancer cell line MDA-MB231, which paved the way for the future use of the system for cancer treatment.