Self-assembly of c-myc DNA promoted by a single enantiomer ruthenium complex as a potential nuclear targeting gene carrier

Abstract

Gene therapy has long been limited in the clinic, due in part to the lack of safety and efficacy of the gene carrier. Herein, a single enantiomer ruthenium(II) complex, Λ-[Ru(bpy)2(p-BEPIP)](ClO4)2 (Λ-RM0627, bpy = 4,4'-bipyridine, p-BEPIP = 2-(4-phenylacetylenephenyl)imidazole [4,5f][1, 10] phenanthroline), has been synthesized and investigated as a potential gene carrier that targets the nucleus. In this report, it is shown that Λ-RM0627 promotes self-assembly of c-myc DNA to form a nanowire structure. Further studies showed that the nano-assembly of c-myc DNA that induced Λ-RM0627 could be efficiently taken up and enriched in the nuclei of HepG2 cells. After treatment of the nano-assembly of c-myc DNA with Λ-RM0627, over-expression of c-myc in HepG2 cells was observed. In summary, Λ-RM0627 played a key role in the transfer and release of c-myc into cells, which strongly indicates Λ-RM0627 as a potent carrier of c-myc DNA that targets the nucleus of tumor cells.

Figure 1. Self-assembly of c-myc G-quadruplex DNA induced by ruthenium complex Λ-RM0627.

(A) Shows the molecular structure of the a single enantiomer ruthenium (II) complex of Λ-RM0627. (B) The electronic spectra of Λ-RM0627 (20 μM, and 3 mL) were determined following the addition of the c-myc G-quadruplex DNA (100 μM) at a rate of 2 μL every 5 minutes. ([c-myc] = 0.0667 μM, n = 0, 1, 2, 3, … 9, etc.) in Tris-HCl buffer (pH 7.2, containing 100 mM KCl). (C) TEM image of free c-myc DNA (5 μM) in Tris–HCl KCl solution (pH 7.2) that was air-dried. (D) TEM image of c-myc DNA (5 μM) self-assembled into nanostructures in the presence of Λ-RM0627 (5 μM) in Tris–HCl KCl buffer (pH 7.2) and air-dried. (E) AFM image of c-myc DNA (5 μM) in the absence of Λ-RM0627. (F) AFM image of the nano-assembly of c-myc DNA (5 μM) in the presence of Λ-RM0627.

Figure 2. The distribution and localization of ruthenium complex Λ-RM0627.

Confocal laser scanning microscopic images of the HepG2 cell nucleus in the presence of Λ-RM0627 (5 μM) treatment for 2 h. The overlay data was analyzed by Image Pro Plus.

Figure 3. The distribution and localization of the nano-assembly.

(A) Confocal laser scanning microscopic image of the HepG2 cellular nucleus in the presence of FAM-c-myc (5 μM) + Λ-RM0627 (5 μM). (B) A 3D-tomoscan image of the HepG2 nucleus in the presence of FITC-c-myc (5 μM) + Λ-RM0627 (5 μM). Red staining shows the a single enantiomer ruthenium (II) complex Λ-RM0627 green staining shows the FAM tagged c-myc G-quadruplex DNA and blue staining shows DAPI nuclear counter-staining. The overlay data was analyzed by Image Pro Plus.

Figure 4. The cellular uptake of the nano-assembly.

(A) Real-time fluorescence images of HepG2 cells treated with c-myc G-quadruplex DNA + Λ-RM0627 (5 μM) for 0, 30, 60, 90, 120, and 150 min. (B) Merged position of green and red fluorescence with time. I_Max is the fluorescence seen at 150 min. (C)Time-dependent cellular uptake of the c-myc G-quadruplex DNA + Λ-RM0627 in HepG2 cells.

Figure 5. Expression of the c-myc gene and the toxicity of the c-myc nano-assembly induced by Λ-RM0627.

(A) Regulation of the expression of c-myc DNA delivered byLipofectamine 2000 and ruthenium complex Λ-RM0627 at the RNA level. [DNA/Lipofectamine 2000] = 1 μg/10 μL; [DNA]: [Λ-RM0627] = 1:1 [DNA] = (0, 1 and 5 μM). (B) The cytotoxicity of the a single enantiomer ruthenium (II) complex Λ-RM0627, free c-myc DNA and Λ-RM0627 +c-myc DNA is shown. All data were obtained from three independent experiments and were presented as the mean ± SD. P < 0.05 vs untreated control. Error bars represent the mean standard deviation.

Figure 6. The potential application of the DNA nano-assembly that was promoted by Λ-RM0627.

Illustration of the c-myc G-quadruplex DNA nano-assembly that was induced by the a single enantiomer ruthenium (II) complex to carry DNA into the nuclei of living cells. Also shown is the potential application of the nano-assembly system as a drug and gene carrier and specific DNA recognizing fluorescent probe.