Uptake and fate of surface modified silica nanoparticles in head and neck squamous cell carcinoma

Abstract

Background: Head and neck squamous cell carcinoma (HNSCC) is currently the eighth leading cause of cancer death worldwide. The often severe side effects, functional impairments and unfavorable cosmetic outcome of conventional therapies for HNSCC have prompted the quest for novel treatment strategies, including the evaluation of nanotechnology to improve e.g. drug delivery and cancer imaging. Although silica nanoparticles hold great promise for biomedical applications, they have not yet been investigated in the context of HNSCC. In the present in-vitro study we thus analyzed the cytotoxicity, uptake and intracellular fate of 200-300 nm core-shell silica nanoparticles encapsulating fluorescent dye tris(bipyridine)ruthenium(II) dichloride with hydroxyl-, aminopropyl- or PEGylated surface modifications (Ru@SiO2-OH, Ru@SiO2-NH2, Ru@SiO2-PEG) in the human HNSCC cell line UMB-SCC 745.

Results: We found that at concentrations of 0.125 mg/ml, none of the nanoparticles used had a statistically significant effect on proliferation rates of UMB-SCC 745. Confocal and transmission electron microscopy showed an intracellular appearance of Ru@SiO2-OH and Ru@SiO2-NH2 within 30 min. They were internalized both as single nanoparticles (presumably via clathrin-coated pits) or in clusters and always localized to cytoplasmic membrane-bounded vesicles. Immunocytochemical co-localization studies indicated that only a fraction of these nanoparticles were transferred to early endosomes, while the majority accumulated in large organelles. Ru@SiO2-OH and Ru@SiO2-NH2 nanoparticles had never been observed to traffic to the lysosomal compartment and were rather propagated at cell division. Intracellular persistence of Ru@SiO2-OH and Ru@SiO2-NH2 was thus traceable over 5 cell passages, but did not result in apparent changes in cell morphology and vitality. In contrast to Ru@SiO2-OH and Ru@SiO2-NH2 uptake of Ru@SiO2-PEG was minimal even after 24 h.

Conclusions: Our study is the first to provide evidence that silica-based nanoparticles may serve as useful tools for the development of novel treatment options in HNSCC. Their long intracellular persistence could be of advantage for e.g. chronic therapeutic modalities. However, their complex endocytotic pathways require further investigations.

Figure 1

Proliferation effects of different surface modified nanoparticles on UMB-SCC 745. BrdU proliferation assays in UMB-SCC 745 cells after incubation (5 h) of nanoparticles with different surface modifications (Ru@SiO₂-OH, Ru@SiO₂-NH₂ and Ru@SiO₂-PEG) at concentration ranges of 0-0.5 mg/ml.

Figure 2

Nanoparticle internalisation. Transmission electron microscopy pictures of nanoparticle internalisation in UMB-SCC 745 exemplarily shown for Ru@SiO₂-NH₂. Uptake occurred either as single nanoparticle (A, B, scale bars = 100 nm), or nanoparticle clusters (C, D, scale bars = 500 nm).

Figure 3

Time dependent uptake of Ru@SiO₂-OH nanoparticles. Ru@SiO₂-OH nanoparticle uptake over 2 h (A and B) and 24 h (C and D) in UMB- SCC 745. A, C: confocal laser scanning microscopy, showing nuclei in blue and Ru@SiO₂-OH nanoparticles in red, scale bars = 20 μm. B, D: transmission electron microscopy, scale bars = 10 μm.

Figure 4

Time dependent uptake of Ru@SiO₂-NH₂ nanoparticles. Ru@SiO₂-NH₂ nanoparticles uptake over 2 h (A and B) and 24 h (C and D) in UMB-SCC 745. A, C: confocal laser scanning microscopy, showing nuclei in blue and Ru@SiO₂-NH₂ nanoparticles in red, scale bars = 20 μm. B, D: transmission electron microscopy, scale bars = 10 μm.

Figure 5

Intracellular localisation of Ru@SiO₂-OH and Ru@SiO₂-NH₂ nanoparticles after 24 h. Transmission electron microscopy showing intracellular localisation of nanoparticles in UMB-SCC 745 after 24 h of incubation. A) Ru@SiO₂-NH₂ nanoparticles and B) Ru@SiO₂-OH nanoparticles. Scale bars = 5 μm.

Figure 6

Time dependent uptake of Ru@SiO₂-PEG nanoparticles. Ru@SiO₂-PEG nanoparticle uptake after 2 h (A and B) and 24 h (C and D) in UMB-SCC 745. A, C: confocal laser scanning microscopy, showing nuclei in blue and Ru@SiO₂-PEG nanoparticles in red, scale bars = 20 μm. B, D: transmission electron microscopy, scale bars = 10 μm.

Figure 7

Ru@SiO₂-OH and Ru@SiO₂-NH₂ nanoparticle uptake in multicellular spheroids. Uptake of nanoparticles in UMB-SCC 745 multicellular spheroids after 5 hours. Confocal laser scanning microscopy pictures, showing Ru@SiO₂-OH (A) and Ru@SiO₂-NH₂ (B) in red and cell nuclei in blue. Scale bars = 100 μm.

Figure 8

Co-localisation of Ru@SiO₂-OH and Ru@SiO₂-NH₂ nanoparticles with early endosomes. Confocal laser scanning microscopy pictures showing a partial co-localisation after 2 h of incubation of Ru@SiO₂-OH (A, in red) or Ru@SiO₂-NH₂ (B, in red) fluorescence with immunosignals for early endosomes protein 1 (A, B, in green). Cell nuclei are stained in blue. Arrows denote large early endosomes, which contain high amounts of nanoparticles. Scale bars = 30 μm.

Figure 9

Intracellular long time retention of nanoparticles. Transmission electron microscopy pictures of UMB-SCC 745 after nanoparticle incubation over a time period of 15 days. A-C) 2, 9 and 12 days after incubation of Ru@SiO₂-OH nanoparticles. D-F) 2, 9 and 12 days after incubation of Ru@SiO₂-NH₂ nanoparticles. Scale bars for A-E) = 5 μm and for F) = 0.5 μm.

Figure 10

Nanoparticle distribution during cell division. Confocal laser scanning microscopy showing distribution of RuSiO₂-NH₂ nanoparticles during cell division (third passage) of UMB-SCC 745. A) metaphase and B) telophase, nucleus in blue and nanoparticles in red. Scale bars = 10 μm.