Cytotoxicity and DNA cleavage with core-shell nanocomposites functionalized by a KH domain DNA binding peptide

Abstract

A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This "KH peptide" enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells.

Figure 1

Schematic representation of experiments performed with the KH-peptide functionalized nanoconjugates.

Figure 2

DNA breakage in the presence of illuminated nanocomposites with differently functionalized surfaces. Lane 1 contains the DNA molecular weight marker; lane 2 is the DNA of the plasmid pGEMII18-WT without addition of any type of nanoparticle. Arrows point to the two forms of DNA: supercoiled DNA indicated by black arrow travels through the gel most rapidly; presence of DNA damaged sites causes plasmid relaxation, this form of DNA, indicated by white arrow, travels through the gel more slowly. Lanes 3 and 4 are pGEMII18-WT plasmid DNA mixed with bare Fe3O4@TiO2 nanoparticles after (3) or before (4) illumination. Lanes 5 and 6 are pGEMII18-WT mixed with Fe3O4@TiO2 nanoparticles covered with ARS only, after or before illumination, respectively. Lanes 7 and 8 are pGEMII18-WT mixed with Fe3O4@TiO2 nanoparticles coated with both KH peptide and Alizarin red S, after (7) or before (8) illumination.

Figure 3

Confocal microscopy images of RPMI 2650 cells treated with nanocomposites and illuminated to activate production of ROS: A, B) control cells in the absence of UV exposure. In control cells 53BP1 is homogenously distributed within the nucleus of the cells, with a solitary focus in some of the cells. C,D) Exposure of control cells to UV light slightly increases the number of 53BP1 foci. E,F) cells treated with ARS covered nanoparticles in the absence of UV exposure, 53BP1 is diffusely distributed within the nucleus without evident nuclear changes. G,H) Cells incubated with non-targeted nanoparticles, covered with ARS only, and exposed to UV light show some 53BP1 foci. I,J) Cells incubated with nanocomposites conjugated to ARS and functionalized with KH-peptide display cytoplasmic as well as nuclear distribution of the nanoparticles. In absence of exposure to a UV light, 53BP1 is homogenously distributed within the nucleus. K,L) UV illumination of cells treated with nanoparticles coated with ARS and KH-peptide lead to development of foci in some of the cells although some cells present only deeply stained small nuclei and shrunken cytoplasm and no staining for 53BP1 (Arrows). (A, C) blue Hoechst staining for DNA and green staining for 53BP1; (B, D) green staining for 53BP1 only; (E, G, I, K) blue Hoechst staining for DNA, green staining for 53BP1 and red staining (ARS) for nanoparticles; (F, H, J, L) green staining for 53BP1 and red staining (ARS) for nanoparticles.

Figure 4

Confocal microscopy of RPMI 2640 cells for detection of apoptosis under the following conditions: A, B) control, untreated cells (; C,D) control cells following exposure to UV light; E, F) cells treated with nanocomposites coated with ARS and functionalized by KH peptide, without exposure to UV light;; G,H) Cells incubated with KH-peptide functionalized nanocomposites and exposed to ultraviolet light show occasional M30 staining (Arrows). (A, C) blue Hoechst staining of DNA; (B, D) green staining with M30 only; (E, G) blue Hoechst staining of DNA; green staining for M30 and red staining for nanoparticles; (F, H) red staining for nanoparticles only.

Figure 5

Flow cytometric analysis of nanocomposite treated cells with or without light activation. Cell staining was done with annexin V (apoptosis positive cells) and SYTOX-blue (necrotic cells). In addition, cellular debris was quantified on the basis of side scatter.