Gold Nanorod-Based Nanohybrids for Combinatorial Therapeutics

Abstract

In this work, multifunctional nanocarriers consisting of poly(sodium-4-styrenesulfonate) (PSS)/doxorubicin (DOXO)/poly-l-lysine hydrobromide (PLL)/hyaluronic acid (HA)-coated and (PSS/DOXO/PLL)₂/HA-coated gold nanorods were assembled by the layer-by-layer technique with the aims of coupling the plasmonic photothermal properties of the metal nanoparticles for plasmonic hyperthermia and the chemoaction of drug DOXO for potential intended combinatorial cancer therapeutics in the future as well as providing different strategies for the controlled and sustained release of the cargo drug molecules. To do that, DOXO could be successfully loaded onto the hybrid nanoconstructs through electrostatic interactions with high efficiencies of up to ca. 78.3 ± 6.9% for the first formed drug layer and 56 ± 13% for the second one, with a total efficiency for the whole system [(PSS/DOXO/PLL)₂/HA-coated NRs] of ca. 65.7 ± 1.4%. Nanohybrid internalization was observed to be enhanced by the outer HA layer, which is able to target the CD44 receptors widely overexpressed in some types of cancers as lung, breast, or ovarian ones. Hence, these nanohybrid systems might be versatile nanoplatforms to simultaneously deliver sufficient heat for therapeutic plasmonic hyperthermia and the anticancer drug. Two controlled mechanisms were proposed to modulate the release of the chemodrug, one by means of the enzymatic degradable character of the PLL layer and another by the modulation of the interactions between the polymeric layers through the exploitation of the optical properties of the hybrid particles under near infrared (NIR) laser irradiation. The combination of this bimodal therapeutic approach exerted a synergistic cytotoxic effect on both HeLa and MDA-MB-231 cancer cells in vitro. Cell death mechanisms were also analyzed, elucidating that plasmonic photothermal therapy induces cell necrosis, whereas DOXO activates the cell apoptotic pathway. Therefore, the present NIR laser-induced targeted cancer thermo/chemotherapy represents a novel targeted anticancer strategy with easy control on demand and suitable therapeutic efficacy.

Scheme 1. Schematic Representation of PSS/DOXO/PLL/HA-Coated (Left) and (PSS/DOXO/PLL)₂/HA-Coated GNRs (Right)

Figure 1

(a) UV–vis absorbance spectra for each PE layer deposited onto GNRs for (a) (PSS/DOXO/PLL)₂/HA-coated GNRs. (b) Longitudinal LSPR shifts (black ●), longitudinal (red ■) and transversal (red □) absorbance decays, and (c) ζ-potentials (black ■) after each coating step for (PSS/DOXO/PLL)₂/HA-coated GNRs. The LSPR shift of coated GNRs with layer i is defined as Δλ = λ(i) – λ(CTAB). Longitudinal and transversal decays are constructed as the ratio between the optical absorbance in layer i regarding that of the CTAB layer: decay(SPR) = abs(i)/abs(CTAB). The lines are only to guide the eye.

Figure 2

Hydrodynamic radii for (a) CTAB-coated (black), PSS-coated (red), PSS/DOXO/PLL-HA-coated (blue), and (PSS/DOXO/PLL)₂-HA-coated (green) GNRs. (b) Scanning TEM image of (PSS/DOXO/PLL)₂/HA-coated GNRs.

Figure 3

DOXO cumulative release profiles at (a) pH 7.4 and (b) pH 5.5 for PSS/DOXO/PLL/HA-coated (squares) and (PSS/DOXO/PLL)₂/HA-coated GNRs (circles) in the absence (black ■, red ●) and presence (black □, red ○) of trypsin. The lines are only to guide the eye. The relative standard deviation (SD) was found less than 10.0%. Error bars were not included for clarity.

Figure 4

NIR-induced DOXO release from (a) PSS/DOXO/PLL/HA-coated and (b) (PSS/DOXO/PLL)₂/HA-coated GNRs. The laser powers were 3.0 (green ▼) and 0.5 (blue ▲) W/cm². Simple drug diffusion (black ■, red ●) and enzymatic-assisted release (black □, red ○) are also shown for comparison. The relative SD was found to be less than 10.0%. Error bars were not included for clarity. The lines are only to guide the eye.

Figure 5

Fluorescence microscopy images of (from top to bottom): bare HeLa cells, bare PSS/PLL/HA-coated GNRs, free DOXO, PSS/DOXO/PLL/HA-coated GNRs, and (PSS/DOXO/PLL)₂/HA-coated GNRs after 4 h of internalization. The blue channel shows the fluorescence from cell nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI; λ_ex = 355 nm); the red channel shows the DOXO fluorescence signal; the TL-BF channel displays the optical direct bright field images; and the merged images show the combination of the three previous channels. Scale bars are 10 μm.

Figure 6

Cellular viability of PSS/DOXO/PLL/HA-coated (black ■), (PSS/DOXO/PLL)₂/HA-coated (red ●), and PSS/PLL/HA-coated GNRs (blue ▲) and free DOXO (green ▼) in (a,b) HeLa and (c,d) MDA-MB-231 cells after 24 and 48 h of incubation.

Figure 7

Photothermal-induced toxicity of PSS/PLL/HA-coated (bare), PSS/DOXO/PLL/HA-coated (monolayer), and (PSS/DOXO/PLL)₂/HA-coated (bilayer) GNRs and free DOXO (DOXO) in (a) HeLa and (b) MDA-MB-231 cells. The cells were irradiated with a continuous-wave (CW) laser at 808 nm at 0 (blue), 0.5 (light blue), 1 (light red), and 3 W/cm² (red) for 5 min. The dotted (50%) and dashed lines (100%) are only to guide the eye.

Figure 8

Ann V/7AAD death cell assay in (a) HeLa and (b) MDA-MB-231 cells. The percentage of cells displaying Annexin V fluorescence is shown in dark orange, and the percentage of necrotic ones stained with 7-ADD is shown in light orange. PSS/DOXO/PLL/HA-coated (monolayer), (PSS/DOXO/PLL)₂/HA-coated (bilayer), and PSS/PLL/HA-coated GNRs were irradiated at several power intensities with a CW NIR laser (808 nm). DOXO was used as a positive control for apoptotic cells. The untreated cells were used as a negative control. The dotted and dashed lines are only to guide the eye.

Figure 9

TEM images of HeLa cells after 6 h of incubation with (a,b) PSS/PLL/HA-coated GNRs and (c,d) (PSS/DOXO/PLL)₂/HA-coated GNRs under irradiation with an 808 nm CW laser for 5 min at 1 W/cm². Scale bars are 2 μm.