Recombinant λ-phage nanobioparticles for tumor therapy in mice models

Lambda phages have considerable potential as gene delivery vehicles due to their genetic tractability, low cost, safety and physical characteristics in comparison to other nanocarriers and gene porters. Little is known concerning lambda phage-mediated gene transfer and expression in mammalian hosts. We therefore performed experiments to evaluate lambda-ZAP bacteriophage-mediated gene transfer and expression in vitro. For this purpose, we constructed recombinant λ-phage nanobioparticles containing a mammalian expression cassette encoding enhanced green fluorescent protein (EGFP) and E7 gene of human papillomavirus type 16 (λ-HPV-16 E7) using Lambda ZAP- CMV XR vector. Four cell lines (COS-7, CHO, TC-1 and HEK-239) were transduced with the nanobioparticles. We also characterized the therapeutic anti-tumor effects of the recombinant λ-HPV-16 E7 phage in C57BL/6 tumor mice model as a cancer vaccine. Obtained results showed that delivery and expression of these genes in fibroblastic cells (COS-7 and CHO) are more efficient than epithelial cells (TC-1 and HEK-239) using these nanobioparticles. Despite the same phage M.O.I entry, the internalizing titers of COS-7 and CHO cells were more than TC-1 and HEK-293 cells, respectively. Mice vaccinated with λ-HPV-16 E7 are able to generate potent therapeutic antitumor effects against challenge with E7- expressing tumor cell line, TC-1 compared to group treated with the wild phage. The results demonstrated that the recombinant λ-phages, due to their capabilities in transducing mammalian cells, can also be considered in design and construction of novel and safe phage-based nanomedicines.     

Optimization of transfection methods for Huh-7 and Vero cells: A comparative study

Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI? and Lipofectamine? 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± 2.36 and 73.9 ± 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI? was 14.2 ± 0.69 and 28 ± 1.11% Huh-7 and Vero cells, respectively. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.