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KMID : 1146920210510020233
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Journal of Pharmaceutical Investigation
2021 Volume.51 No. 2 p.233 ~ p.242
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Treatment of Wilms’ nephroblastoma cancer cells via EGFR targeting of dactinomycin loaded DNA-nanowires
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Baig Mirza Muhammad Faran Ashraf
Zhang Chengfei
Akhtar Muhammad Furqan
Saleem Ammara
Nisar Naveed
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Abstract
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Purpose: 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.
Methods: 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.
Results: 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.
Conclusion: 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.
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KEYWORD
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Dactinomycin (DCTM), DNA-nanowires (D-NWs), Epidermal growth factor (EGF), EGFR (receptors), Resistant cancer
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