Self-Targeting of Carbon Dots into the Cell Nucleus: Diverse Mechanisms of Toxicity in NIH/3T3 and L929 Cells

Abstract

It is important to understand the nanomaterials intracellular trafficking and distribution and investigate their targeting into the nuclear area in the living cells. In our previous study, we firstly observed penetration of nonmodified positively charged carbon dots decorated with quaternary ammonium groups (QCDs) into the nucleus of mouse NIH/3T3 fibroblasts. Thus, in this work, we focused on deeper study of QCDs distribution inside two healthy mouse NIH/3T3 and L929 cell lines by fluorescence microspectroscopy and performed a comprehensive cytotoxic and DNA damage measurements. Real-time penetration of QCDs across the plasma cell membrane was recorded, concentration dependent uptake was determined and endocytic pathways were characterized. We found out that the QCDs concentration of 200 µg/mL is close to saturation and subsequently, NIH/3T3 had a different cell cycle profile, however, no significant changes in viability (not even in the case with QCDs in the nuclei) and DNA damage. In the case of L929, the presence of QCDs in the nucleus evoked a cellular death. Intranuclear environment of NIH/3T3 cells affected fluorescent properties of QCDs and evoked fluorescence blue shifts. Studying the intracellular interactions with CDs is essential for development of future applications such as DNA sensing, because CDs as DNA probes have not yet been developed.

Keywords: L929; NIH/3T3; carbon dots; cnucleus; cytotoxicity; fibroblasts; fluorescence microspectroscopy; genotoxicity; nucleolus.

Figure 1

Fluorescent imaging of NIH/3T3 cells exposed to QCDs for 24 h: (a) 50 µg/mL; (b) 100 µg/mL; (c) 200 µg/mL; (d) 400 µg/mL; “Nu” and arrows denotes the nuclei. FMS, field of view 91 µm.

Figure 2

(a) Viability and (b) genotoxicity of NIH/3T3 cells exposed to the concentration line of QCDs and incubated for 24 h. Please note the following genotoxicity terms: “head” is intact DNA in nucleus, “tail” is damaged DNA migrated away from the nucleus; when the tail value is more than 10%, the dose is genotoxic.

Figure 3

The nuclei of NIH/3T3 cells filled with QCDs. (a) Fluorescence intensity image generated by summing up all the images in the λ-stack (FMS, field of view 132 µm); (b) False colored maps of intensity; (c) Spectral peak position obtained from spectral fitting. Concentration of QCDs was 400 µg/mL with an incubation time of 24 h.

Figure 4

Detailed image of the nucleus filled with QCDs (400 µg/mL, 24 h, washed in PBS) shown in blue color and nucleus membrane represented by a violet ring. NIH/3T3 cells; field of view of 55 µm.

Figure 5

Fluorescent imaging of L929 cells exposed to QCDs for 24 h: (a) 50 µg/mL; (b) 100 µg/mL; (c) 200 µg/mL; (d) 400 µg/mL—dead cell with QCDs inside the nucleus and nucleoli; “Nu” and arrows denotes the nuclei. FMS, field of view 91 µm.

Figure 6

(a) Viability and (b) genotoxicity of L929 cells exposed to the concentration line of QCDs and incubated 24 h. Please note the following genotoxicity terms: “head” is intact DNA in nucleus, “tail” is damaged DNA migrated away from the nucleus; when the tail value is more than 10%, the dose is genotoxic.

Figure 7

Concentration dependent uptake and endocytosis of both cell lines: blue and violet lines—NIH/3T3, L929 cells incubated in standard conditions; orange and pink lines—NIH/3T3, L929 cells labeled after inhibition of endocytosis.

Figure 8

Inhibition of different endocytic mechanisms in L929 and NIH/3T3 cells immediately after adding the QCDs concentration of 200 µg/mL; incubation time of inhibitors was 2 h.

Figure 9

Cell cycle profile of cells labeled with QCDs: (a) Healthy mouse NIH/3T3 fibroblasts, (b) Healthy mouse L929 fibroblasts.

Figure 10

Spectral fitting of local intensities and emission spectra maximum wavelengths.