Size-dependent localization and penetration of ultrasmall gold nanoparticles in cancer cells, multicellular spheroids, and tumors in vivo

Abstract

This work demonstrated that ultrasmall gold nanoparticles (AuNPs) smaller than 10 nm display unique advantages over nanoparticles larger than 10 nm in terms of localization to, and penetration of, breast cancer cells, multicellular tumor spheroids, and tumors in mice. Au@tiopronin nanoparticles that have tunable sizes from 2 to 15 nm with identical surface coatings of tiopronin and charge were successfully prepared. For monolayer cells, the smaller the Au@tiopronin NPs, the more AuNPs found in each cell. In addition, the accumulation of Au NPs in the ex vivo tumor model was size-dependent: smaller AuNPs were able to penetrate deeply into tumor spheroids, whereas 15 nm nanoparticles were not. Owing to their ultrasmall nanostructure, 2 and 6 nm nanoparticles showed high levels of accumulation in tumor tissue in mice after a single intravenous injection. Surprisingly, both 2 and 6 nm Au@tiopronin nanoparticles were distributed throughout the cytoplasm and nucleus of cancer cells in vitro and in vivo, whereas 15 nm Au@tiopronin nanoparticles were found only in the cytoplasm, where they formed aggregates. The ex vivo multicellular spheroid proved to be a good model to simulate in vivo tumor tissue and evaluate nanoparticle penetration behavior. This work gives important insights into the design and functionalization of nanoparticles to achieve high levels of accumulation in tumors.

Figure 1

Characterization of 2, 6, and 15 nm gold nanoparticles with the same surface modification. (a) TEM images and corresponding size measurements of synthesized tiopronin coated Au nanopartices (Au@tiopronin NPs). Scale bars are 100 nm; the middle panel contains an additional scale bar of 20 nm. (b) Zeta-potential of the nanoparticles ranging from −32 to −41 eV. (c) Spectrum of the nanoparticles at wavelengths between 400 and 800 nm and corresponding photographs of the Au@tiopronin NPs dispersed in pure water. (d) XRD patterns of the synthesized Au@tiopronin NPs. (e) XPS graphs of the Au@tiopronin NPs.

Figure 2

Uptake of 2, 6, and 15 nm AuNPs by MCF-7 monolayer cells. (a) Bright field images of cells after treatment with 1 nM gold nanoparticles for 24 h. (b) Quantitative ICP-MS measurement of AuNP uptake by cells treated as in (a). (c) Percentage of gold nanoparticles localized in the nucleus compared to the whole cell after treatment with 2 and 6 nm Au nanoparticles at 1 nM. (d) TEM images of cells treated with 1 nM nanoparticles for 24 h. Red arrows indicate the gold nanoparticles. Boxed regions are enlarged in the adjacent panels.

Figure 3

Characterization of MCF-7 monolayers and multicellular spheroids. (a) Bio-SEM of monolayer cells and 7-day-old spheroids seeded at a concentration of 600 cells per well. (b) Flow cytometry analysis of cell populations within 7- and 14-day-old spheroids seeded at a concentration of 600 cells per well. (c) Images of 7- and 14-day-old spheroids after HE staining. Apoptotic cells are located within the green circle, while proliferative cells are located between the green circle and the red circle. (d) TEM images of MCF-7 monolayer cells and of cells in the outer and inner regions of 14-day-old spheroids.

Figure 4

Penetration of 1 nM AuNPs into tumor spheroids after treatment for 3 and 24 h. (a) Images of spheroids treated with nanoparticles of different sizes after HE staining. (b) ICP-MS analysis of the number of Au nanoparticles in each treated spheroid. (c) Dark field images of spheroids after culture with nanoparticles of different sizes. (d) Representative TEM images of the external and internal regions of spheroids treated for 24 h. Red arrows indicate the gold nanoparticles. (e) Schematic illustration of the penetration behavior of AuNPs of different sizes.

Figure 5

Pharmacokinetics and biodistribution of 2, 6, and 15 nm AuNPs. (a) Blood elimination profiles of Au following a single intravenous injection of Au nanoparticles at a dose of 5 mg Au/kg in tumor-bearing mice. Data represent mean ± SD (n = 3). (b) Au content in tumor, heart, liver, spleen, lung, brain, and kidney 24 h after iv injections of gold nanoparticles at 5 mg Au/kg. Data represent mean ± SD (n = 3) (c) Representative TEM micrographs of tumor tissue taken 24 h after the administration of AuNPs.