Modulation of the Microtubule Network for Optimization of Nanoparticle Dynamics for the Advancement of Cancer Nanomedicine

Abstract

Nanoparticles (NPs) have shown promise in both radiotherapy and chemotherapy. NPs are mainly transported along cellular microtubules (MTs). Docetaxel (DTX) is a commonly used chemotherapeutic drug that can manipulate the cellular MT network to maximize its clinical benefit. However, the effect of DTX on NP behaviour has not yet been fully elucidated. We used gold NPs of diameters 15 and 50 nm at a concentration of 0.2 nM to investigate the size dependence of NP behaviour. Meanwhile, DTX concentrations of 0, 10 and 50 nM were used to uphold clinical relevance. Our study reveals that a concentration of 50 nM DTX increased NP uptake by ~50% and their retention by ~90% compared to cells treated with 0 and 10 nM DTX. Smaller NPs had a 20-fold higher uptake in cells treated with 50 nM DTX vs. 0 and 10 nM DTX. With the treatment of 50 nm DTX, the cells became more spherical in shape, and NPs were redistributed closer to the nucleus. A significant increase in NP uptake and retention along with their intracellular distribution closer to the nucleus with 50 nM DTX could be exploited to target a higher dose to the most important target, the nucleus in both radiotherapy and chemotherapy.

Keywords: cancer; docetaxel; microtubules; nanomedicine; nanoparticles.

Figure 1

Intracellular transport of NPs. (a) Schematic diagram showing the transport of vesicles containing NPs within the cellular microtubule network. MTs are long tubulin polymers and are often anchored at the centrosome. Their plus ends extend toward the cell periphery, whereas their minus ends are located closer to the cell centre and are often anchored at the centrosome. Inset figure: Vesicular transport along MTs is supported by the motor proteins, dynein and kinesin. NP transport along actin filaments is supported by motor proteins closer to the cell periphery. (b) Representation of a single GNP functionalized with PEG and RGD peptides. (c) Snapshot of a live cell showing vesicles containing NPs (marked in red; left most), MTs (marked in green; middle), and the merged image (right most). Scale bar is 20 µm.

Figure 2

The effect of NP size and DTX concentration on intracellular NP accumulation. (a) Schematic diagram highlighting differences in the MT network and NP distribution in the absence and presence of DTX. (b) Size-dependent uptake of NPs in MDA-MB-231 and HeLa cells. (c) Size-dependent uptake of NPs in the presence of a 50 nm DTX concentration in MDA-MB-231 cells. (d) DTX concentration dependent NP uptake in MDA-MB-231 and HeLa cells. (e–g) Images showing NPs and the MT network in cells treated with 0, 10, and 50 nM of DTX, respectively (f): arrow indicates cell fragmentation during division; g: arrows indicate bundling of MTs). (h–i) Z-stack showing the distribution of NPs and the MT network in a group of cells treated with 0 and 50 nM DTX, respectively. Vesicles containing GNPs and MTs are marked in red and green, respectively. Scale bar is 20 µm. Error bars are standard deviations from 3 replicate measurements. * Represents a statistically significant difference (Welch’s unequal variance t-test, p < 0.05).

Figure 3

Distribution of NPs during cell division in the presence of DTX. (a) Schematic diagram illustrating the different outcomes of cell division in the presence of the antimitotic drug, DTX. (b) Cell cycle analysis for control cells and cells treated with 10 and 50 nM concentrations of DTX. (c) Distribution of NPs in a control cell (c-1), cells treated with 50 nM DTX (c-2–c-4) and in a cell treated with 10 nM DTX (c-5). Vesicles containing GNPs and MTs are marked in red and green, respectively. Scale bars are 20 µm.

Figure 4

Retention of NPs in cells treated with DTX. (a) Retention of NPs in DTX-treated cells after 24 h. (b) Cell cycle analysis for control HeLa cells and cells treated with 50 nM DTX at the beginning and end of the retention. (c) NP size-dependent retention in MDA-MB-231 cells treated with 50 nM DTX. (d,e) Images corresponding to control cells and cells treated with 50 nM DTX after 24 h, respectively. (f,g) Z-stack showing the distribution of NPs in different planes following the retention process in a control cell and a cell treated with 50 nM DTX, respectively. Vesicles containing GNPs and MTs are marked in red and green, respectively. Scale bar is 20 µm. Error bars are standard deviations from 3 replicate measurements. * Represents a statistically significant difference (Welch’s unequal variance t-test, p < 0.05).

Figure 5

Action of DTX in the presence of GNPs. (a,b) The biocompatibility of GNPs was verified by measuring the cell survival fraction and DNA damage, respectively. (b,c) The action of the drug in the presence of GNPs was verified by measuring the DNA damage and cell survival fraction, respectively. (d) Mapping of DNA double strand breaks (DSBs) in control cells and cell treated with 50 nM DTX. DNA DSBs and nuclei are marked in green and blue, respectively. Scale bar is 20 µm. Error bars are standard deviations from 3 replicate measurements. * Represents a statistically significant difference (Welch’s unequal variance t-test, p < 0.05).

Figure 6

The relative timing of DTX and GNP inoculation in MDA-MB-231 cells treated with 0 and 50 nM DTX. (a) Variation in intracellular GNP accumulation with simultaneous addition of GNP and DTX vs. GNPs added 6 hrs after the addition of DTX. (b) Cell cycle analysis for control cells and cells treated with 50 nM DTX after 4, 8, and 24 h. (c,d) Images of individual cells treated with 0 and 50 nM DTX, respectively. Vesicles containing GNPs and MTs are marked in red and green, respectively. Scale bar is 20 µm. Error bars are standard deviations from 3 replicate measurements. * Represents a statistically significant difference (Welch’s unequal variance t-test, p < 0.05).