Treatment of Wilms’ nephroblastoma cancer cells via EGFR targeting of dactinomycin loaded DNA-nanowires

Abstract

Dactinomycin (DCTM) is a highly cytotoxic hydrophobic drug requiring robust nanomaterials for uniformed water dispersion and safe delivery to tumor site avoiding exposure to healthy cells. DNA triangulation produces sturdier two-dimensional nanostructures through the polymerization of DNA-triangles by sticky ends cohesion in the form of DNA-nanosheets. The curvature of the B-form (right twisted) DNA causes the coiling of the DNA-nanosheets into DNA-nanowires (D-NWs) structures. DNA-triangles scaffolded by the short circular templates (84-NT) are stiffer in topology giving rise to compact D-NWs for DCTM loading, and cellular delivery. The PAGE gel analysis was performed to assess the polymerization of the DNA-triangles to observe restricted electrophoretic mobility, and attainment of a single sharp band. The morphology and compactness of the D-NWs were confirmed by the AFM analysis and confocal imaging. Epidermal growth factor (EGF) functionalization of the D-NWs was performed through amide chemistry using amino-modified DNA strands reacting with the carboxylic group of EGF for EGFR targeting. EGFR is highly expressed on NB-OK-1 Wilms’ tumor nephroblastoma cancer cells. DCTM loading onto D-NWs was carried out through intercalation between the base pairs of GC rich DNA duplex by physical mixing/incubation, and was confirmed through the UV peak shift analysis and confocal imaging. Cell internalizations and the cytotoxic effects were monitored via confocal imaging, MTT assay, and flow cytometry. AFM images of the synthesized D-NWs showed that polymerization of DNA-triangles was successful with the length ranging from 4 to 6 µm, and width ranging from 80 to 120 nm. EGF functionalization was confirmed through the confocal microscopy after labeling EGF with the FITC hook conjugating dye. The slight UV shift (> 15 nm) confirmed DCTM loading onto D-NWs. Blank D-NWs showed biocompatibility to the cells at different (low to high) concentrations (10 µM to 640 µM). MTT assay revealed that DCTM loaded D-NWs showed a dose-dependent (0.25–128 nM) decrease in cell viability. EGF functionalized D-NWs effectively targeted the EGFR rich NB-OK-1 cancer cells compared to the control HEK293/D75 cells lacking EGFR (receptors). By these results, we can expect similar site-specific targeted treatment if administered systemically.