Recombinant lambda-phage nanobioparticles for tumor therapy in mice models

Abstract

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 lambda-phage nanobioparticles containing a mammalian expression cassette encoding enhanced green fluorescent protein (EGFP) and E7 gene of human papillomavirus type 16 (lambda-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 lambda-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 lambda-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 lambda-phages, due to their capabilities in transducing mammalian cells, can also be considered in design and construction of novel and safe phage-based nanomedicines.

Figure 1

A) Presence of EGFP gene (700 bp) in packaged phages was confirmed using PCR. Lane 1 is negative control. Lane 2 is the result of PCR. Lane 3 is gene ruler from Fermentaz. B) Plaque formation by recombinant λ phages on top agar.

Figure 2

Packaging efficiency of wild-λ-DNA, pBR322-λ-DNA, EGFP-λ-DNA, and E7-λ-DNA before (series 2) and after (series 1) packaging using Gigapack.

Figure 3

A. CHO cells transfected by EGFP-λ-phage nanobioparticles using fluorescent microscopy. Four cell types COS-7, CHO, TC-1 and HEK-293 were transfected by EGFP-λ-phages. The best GFP expression was observed after 48 hours by fluorescent microscopy 48 hours later in CHO cells. B. CHO experimental control.

Figure 4

SDS-PAGE and Western blot analyses of CHO cells infected with E7-λ-phages. After overnight incubation, the cellular proteins were extracted and analyzed by SDS-PAGE and immunoblotting.

Figure 5

The internalizing analyses for four cell lines (COS-7, TC-1, HEK-293, and CHO).

Figure 6

Therapeutic vaccination against TC-1-induced tumors. Mice were inoculated with 2 × 105 TC-1 tumor into the right flank and then treated with recombinant λ-ZAP E7 phage, Wild λ-ZAP phage (phage control) and PBS (negative control) 7 days after inoculation. Mice were monitored for evidence of tumor growth by palpation and inspection twice a week. For determining of tumor volume, each individual tumor size was measured. Line and scatter plot graphs depicting the tumor volume (mm3) are presented. The data presented is a representation of two independent experiments.