Intercellular Trafficking of Gold Nanostars in Uveal Melanoma Cells for Plasmonic Photothermal Therapy

Abstract

Efficient plasmonic photothermal therapies (PPTTs) using non-harmful pulse laser irradiation at the near-infrared (NIR) are a highly sought goal in nanomedicine. These therapies rely on the use of plasmonic nanostructures to kill cancer cells while minimizing the applied laser power density. Cancer cells have an unsettled capacity to uptake, retain, release, and re-uptake gold nanoparticles, thus offering enormous versatility for research. In this work, we have studied such cell capabilities for nanoparticle trafficking and its impact on the effect of photothermal treatments. As our model system, we chose uveal (eye) melanoma cells, since laser-assisted eye surgery is routinely used to treat glaucoma and cataracts, or vision correction in refractive surgery. As nanostructure, we selected gold nanostars (Au NSs) due to their high photothermal efficiency at the near-infrared (NIR) region of the electromagnetic spectrum. We first investigated the photothermal effect on the basis of the dilution of Au NSs induced by cell division. Using this approach, we obtained high PPTT efficiency after several cell division cycles at an initial low Au NS concentration (pM regime). Subsequently, we evaluated the photothermal effect on account of cell division upon mixing Au NS-loaded and non-loaded cells. Upon such mixing, we observed trafficking of Au NSs between loaded and non-loaded cells, thus achieving effective PPTT after several division cycles under low irradiation conditions (below the maximum permissible exposure threshold of skin). Our study reveals the ability of uveal melanoma cells to release and re-uptake Au NSs that maintain their plasmonic photothermal properties throughout several cell division cycles and re-uptake. This approach may be readily extrapolated to real tissue and even to treat in situ the eye tumor itself. We believe that our method can potentially be used as co-therapy to disperse plasmonic gold nanostructures across affected tissues, thus increasing the effectiveness of classic PPTT.

Keywords: femtosecond pulse laser; gold nanostars; nanoparticle endocytosis; nanoparticle exocytosis; plasmonic photothermal therapy.

Figure 1

(a) TEM image of the synthetized Au NSs. (b) UV-vis-near-infrared (NIR) spectra of the gold nanostars (Au NS) colloidal solution with the localized surface plasmon resonance (LSPR) band at ca. 800 nm (red) and at 795 nm (blue), before and after transfer to physiological conditions, respectively. (c) Cell viability test after 12 h of incubation of Au NSs with uvea cells (3 independent samples were tested and averaged; the error bars show the standard deviation of the relative cell viability). The cell viability shows cell survival above 90% after 19 days.

Figure 2

(a) Approach one: 1. Incubation of Au NSs for 12 h with uveal melanoma cells. 2. When the culture reached 95% confluence, the cells were divided in two different stocks. 3. The first stock with 50% of the cells was used for laser experiments. 4. The second stock with the other 50% of the cells was left in culture until 95% confluence, at which point it was again divided in two equal stocks to repeat the process. (b) Fresh cells and Au NS-loaded cells (8 pM) were mixed at a 1:1 ratio (i.e., Au NS effective concentration of ~4 pM). When the culture reached 95% confluence, it was split in two as explained above (steps 3 and 4) and the process was repeated five times.

Figure 3

(a) TEM micrograph of a single cell loaded with Au NSs and (b) image at higher magnification after incubation with 8 pM Au NSs. (c) Micrograph after 48 h of incubation and (d) image at higher magnification, in which many regions can be seen where the Au NSs have been exocyted and locate between two cells, as well as small vesicular compartments with smaller amounts of Au NS.

Figure 4

Cell viability as a function of the applied fs laser power density (3 independent samples were tested and averaged at each power density; the error bars show the standard deviation of the relative cell viability). (a) After incubation with Au NSs, we observed an important killing rate using laser power densities above 0.21 W/cm². Similar results were obtained after the first (b), second (c), third (d), and fourth (e) cell passage, with small differences of 10–20% of cell survival. After the fifth passage (f), a laser power density of at least 0.42 W/cm² was required to obtain significant cell killing rates.

Figure 5

Cell viability as a function of the applied fs laser power density (3 independent samples were tested and averaged at each power density; the error bars show the standard deviation of the relative cell viability). (a) After mixing 50% of Au NS-loaded cells and 50% of non-loaded cells, we observed a killing rate of ca. 50% (see blue dashed line) using laser power densities above 0.21 W/cm². After the first (b), second (c), third (d), and fourth (e) cell passage, the cell killing rate increased with small differences (10–20%) in cell survival using a laser power density of at least 0.21 W/cm². After the fifth passage (f), we did not observe important cell killing rates.

Figure 6

(a) TEM micrograph of a single cell where a big membrane invagination can be observed and (b) image at higher magnification. Another example of the exocytosis and re-endocytosis of Au NSs is shown in (c) and at higher magnification in (d).