Cu-Hemin Nanosheets and Indocyanine Green Co-Loaded Hydrogel for Photothermal Therapy and Amplified Photodynamic Therapy

Abstract

Near-infrared (NIR) organic small molecule indocyanine green (ICG) could respond well to 808 nm laser to promote local high temperature and ROS generation for realizing photothermal therapy (PTT)/photodynamic therapy (PDT). However, the high content of GSH in the tumor microenvironment (TME) limited the further therapeutic performance of ICG. Herein, injectable agarose in situ forming NIR-responsive hydrogels (CIH) incorporating Cu-Hemin and ICG were prepared for the first time. When CIH system was located to the tumor tissue through local injection, the ICG in the hydrogel could efficiently convert the light energy emitted by the 808 nm laser into thermal energy, resulting in the heating and softening of the hydrogel matrix, which releases the Cu-Hemin. Then, the over-expressed GSH in the TME could also down-regulated by Cu-Hemin, which amplified ICG-mediated PDT. In vivo experiments validated that ICG-based PDT/PTT and Cu-Hemin-mediated glutathione depletion could eliminate cancer tissues with admirable safety. This hydrogel-based GSH-depletion strategy is instructive to improve the objective response rate of PDT.

Keywords: glutathione; hydrogel; indocyanine green; photodynamic therapy; photothermal therapy.

Scheme 1

Schematic illustration of Cu-Hemin nanosheets and indocyanine green co-loaded hydrogel for photothermal therapy and amplified photodynamic therapy.

Figure 1

Characterization analysis of Cu-Hemin. (A) TEM image of Cu-Hemin nanosheets. (B) XRD pattern of Cu-Hemin. (C) The XPS spectrum of Cu-Hemin. (D) The Cu 2p XPS spectra of Cu-Hemin nanosheets. (E) The relative GSH content of the supernatant after the reaction of various concentrations Cu-hemin and 10 mm GSH in mixed solution for 24 h.

Figure 2

Characterization analysis of ICG. (A) The prepared image of pure hydrogel (i) and CIH (ii). (B) Rheological and temperature curves for the prepared CIH. (C) The corresponding thermal images and (E) photothermal heating curves of CIH at different concentrations (ICG: 0, 20 and 100 μg/mL) under an 808 nm (NIR-I) laser irradiation at a power density of 0.5 W/cm. (D) Absorption spectrum of ICG solution.

Figure 3

Digital image analysis of softening effect of CIH. (A) The morphology of the prepared CIH before and after 0.5 W/cm² 808 nm laser irradiation for 5 min. (B) The infrared thermal images of the prepared CIH before and following irradiation. (C) Relevant 3D temperature diagram in 2B.

Figure 4

In vitro experiments. (A) Fluorescence images of 4T1 cells stained by DCFH-DA to indicate nanoparticle-induced ROS generation. Scar bar: 50 μm. (B) Corresponding quantitative analysis of ROS generation in 4A. (C) Viability of 4T1 cells cultured in the presence of various formulations. (D) Cell viability of 4T1 cells following the CIH treatments under different ICG concentration. **P < 0.01; Student’s t-test.

Figure 5

In vivo experiments. (A) Relative changes of tumor volume in mice bearing 4T1 tumors after indicated treatments. (B) Body weight changes of treated mice. (C) Tumor weight measured following the indicated treatments. (D) Representative digital photos of tumors collected from various groups. (D) TUNEL and Ki-67 tumor sections from the indicated treatment groups. **P < 0.01; Student’s t-test.