Loading of Indocyanine Green within Polydopamine-Coated Laponite Nanodisks for Targeted Cancer Photothermal and Photodynamic Therapy

Abstract

The combination of photothermal therapy (PTT) and photodynamic therapy (PDT) in cancer treatment has attracted much attention in recent years. However, developing highly efficient and targeted therapeutic nanoagents for amplifying PTT and PDT treatments remains challenging. In this work, we developed a novel photothermal and photodynamic therapeutic nanoplatform for treatment of cancer cells overexpressing integrin α_vβ₃ through the coating of polydopamine (PDA) on indocyanine green (ICG)-loaded laponite (LAP) and then further conjugating polyethylene glycol-arginine-glycine-aspartic acid (PEG-RGD) as targeted agents on the surface. The ICG/LAP⁻PDA⁻PEG⁻RGD (ILPR) nanoparticles (NPs) formed could load ICG with a high encapsulation efficiency of 94.1%, improve the photostability of loaded ICG dramatically via the protection of PDA and LAP, and display excellent colloidal stability and biocompatibility due to the PEGylation. Under near-infrared (NIR) laser irradiation, the ILPR NPs could exert enhanced photothermal conversion reproducibly and generate reactive oxygen species (ROS) efficiently. More importantly, in vitro experiments proved that ILPR NPs could specifically target cancer cells overexpressing integrin α_vβ₃, enhance cellular uptake due to RGD-mediated targeting, and exert improved photothermal and photodynamic killing efficiency against targeted cells under NIR laser irradiation. Therefore, ILPR may be used as effective therapeutic nanoagents with enhanced photothermal conversion performance and ROS generating ability for targeted PTT and PDT treatment of cancer cells with integrin α_vβ₃ overexpressed.

Keywords: indocyanine green; laponite; photodynamic therapy; photothermal therapy; polydopamine.

Scheme 1

Schematic illustration of the synthesis of ICG/LAP-PDA-mPEG and ICG/LAP-PDA-PEG-RGD (ILPR) nanoparticles (NPs).

Figure 1

(a) Ultraviolet/visible (UV-vis) spectra of LAP, ICG and ICG/LAP; (b) X-ray diffraction (XRD) patterns of LAP, ICG and ICG/LAP; (c) temperature rising curve of water, LAP, ICG, and ICG/LAP solutions; and (d) temperature changes of ICG and ICG/LAP solutions at the same ICG concentration (C_ICG = 120 μg/mL) under an 808 nm laser irradiation (1.2 W/cm²) for 3 cycles (3 min of irradiation for each cycle).

Figure 2

(a) ¹H nuclear magnetic resonance (NMR) spectra in D₂O and (b) Fourier transform-infrared (FT-IR) spectra of LAP, PDA and LAP–PDA; (c) TGA curves of LAP and LAP-PDA; (d) temperature rising curves of LAP and LAP–PDA solutions (C_PDA = 300 μg/mL) under an 808 nm laser irradiation (1.2 W/cm², 3 min), respectively.

Figure 3

(a) UV-vis spectra of ICG, LAP, ICG/LAP, LAP–PDA and ICG/LAP–PDA at the same ICG concentration; (b) temperature rising curveof water, LAP, ICG, ICG/LAP, LAP–PDA and ICG/LAP–PDA solutions at the same ICG concentration (C_ICG = 100 μg/mL) under an 808 nm laser irradiation (1.2 W/cm², 3 min);(c) temperature changes of free ICG, ICG/LAP, LAP–PDA and ICG/LAP–PDA solutions at the same ICG concentration (C_ICG = 100 μg/mL) under irradiation of the 808 nm laser for 5 cycles (1.2 W/cm², 3 min of irradiation for each cycle); (d) ICG release from ICG/LAP and ICG/LAP–PDA at 37 °C in the acetate buffers (pH = 5.0).

Figure 4

(a) The transmission electron microscope (TEM) image and (b) corresponding size distribution of ICG/LAP–PDA–mPEG; (c)the TGA curves of LAP, ICG/LAP, ICG/LAP–PDA, ICG/LAP–PDA–mPEG and ILPR, respectively; (d) temperature rising curve of ICG/LAP–PDA, ICG/LAP–PDA–mPEG and ILPR solutions (C_ICG = 100 μg/mL) under an 808 nm laser irradiation (1.2 W/cm², 3 min).

Figure 5

(a) CCK-8 viability assay of MDA-MB-231 cells after treatment with LAP–PDA–PEG–RGD, ICG/LAP–PDA–mPEG and ILPR NPs at the same ICG concentration (C_ICG = 5, 10, 20, 30, 40 μg/mL) for 24 h, respectively; (b) Cellular uptake of Si of MDA-MB-231 cells after treatment with ICG/LAP–PDA–mPEG and ILPR NPs at the same ICG concentration (C_ICG=5, 10, 20, 30, 40 μg/mL) for 6 h, respectively. Phosphate-buffered saline(PBS) buffer was used as control. One-way ANOVA statistical analysis was performed to evaluate the experimental data. A p value of 0.05 was selected as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively.

Figure 6

(a) Consumption of DPBF (1,3-diphenylisobenzofuran)over time due to ¹O₂ generation for water, LAP, ICG, LAP–PDA–PEG–RGD and ILPR aqueous solution with an 808 nm laser irradiation (1.2 W/cm²); (b) mean fluorescence of DCF in MDA-MB-231cells stained by DCF-H after incubated with LAP–PDA–PEG–RGD and ILPR NPs at the same ICG concentration (C_ICG = 10, 40 μg/mL) with/without laser irradiation (1.2 W/cm², 5 min), a p value of 0.05 was selected as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively; (c) fluorescence microscopic images of MDA-MB-231cells stained by DCF-H after the cells incubated with LAP–PDA–PEG–RGD and ILPR NPs (C_ICG = 40 μg/mL) with/without laser irradiation (+L/-L) (1.2 W/cm², 5 min).PBS buffer was used as control.

Figure 7

Cell viabilities of MDA-MB-231 cells after incubated with LAP–PDA–PEG–RGD, ICG/LAP–PDA–mPEG and ILPR NPs (a) at different ICG concentrations (C_ICG = 10, 20, 40 μg/mL) with/without irradiation of an 808 nm laser (2.5 cm², 1.2 W/cm², 5 min); and (b) at the same ICG concentration (C_ICG = 40 μg/mL) with an 808 nm laser irradiation (2.5 cm², 5 min) at different power densities (0.8, 1.0, 1.2 W/cm²), ap value of 0.05 was selected as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively.; (c) fluorescence microscopic images of Calcein AM and PI co-staining MDA-MB-231 cells after treatment with PBS, LAP–PDA–PEG–RGD, ICG/LAP–PDA–mPEG and ILPR at the same ICG concentration (C_ICG = 40 μg/mL) with/without (−L/+L) irradiation of an 808 nm laser (0.25 cm², 1.2 W/cm², 5 min). PBS buffer was used as control.