Synergistic photothermal/photodynamic suppression of prostatic carcinoma by targeted biodegradable MnO2 nanosheets

Abstract

The biodegradability and clearance of metal-based nanomaterials have been questioned worldwide, which have greatly limited their clinical translation. Herein, ultrathin manganese dioxide (MnO₂) nanosheets with broad near-infrared (NIR) absorption and pH-dependent degradation properties were prepared. After being modified with polyethylene glycol-cyclic arginine-glycineaspartic acid tripeptide (PEG-cRGD), the MnO₂ nanosheets were then used as photothermal agent and nanocarrier to encapsulate chlorin e6 (Ce6) for targeted photothermal (PTT) and photodynamic (PDT) of cancer. As expected, the MnO₂-PEG-cRGD nanosheets show high Ce6 loading capacity (351 mg/g), superb photothermal conversion performance (37.2%) and excellent colloidal stability. These nanosheets also exhibit pH-dependent and NIR-induced Ce6 release. Furthermore, the MnO₂ nanosheets can be degraded by reacting with hydrogen peroxide in the acidic microenvironment, which are able to elevate the oxygen concentration in situ and thus reverses the tumor hypoxia. Thanks to these favorable properties and the cRGD-mediated tumor-targeted ability, the fabricated MnO₂-PEG-cRGD/Ce6 nanocomposites can be effectively up taken by alpha-v beta-3 (α_vβ₃) integrin over-expressed prostatic carcinoma PC3 cells and achieve favorable therapeutic outcomes under a single 660 nm NIR laser, which is also verified by in vitro studies. The biodegradable MnO₂-PEG-cRGD/Ce6 nanosheets developed in this work can be a promising nanoplatform for synergetic PTT/PDT cancer therapy.

Keywords: Ce6; MnO nanosheet; photodynamic therapy; photothermal therapy; targeted delivery.

Scheme 1.

Schematic illustration for the preparation of MnO₂-PEG-cRGD/Ce6 composite as a biodegradable nanoplatform for synergistic PTT/PDT targeted therapy.

Figure 1.

TEM images of (A) MnO₂ and (B) MnO₂-PEG-cRGD nanosheets. (C) DLS size distribution of MnO₂ and MnO₂-PEG-cRGD nanosheets. (D) Mn 2p XPS spectra for MnO₂ nanosheets. (E) Zeta potential of MnO₂, MnO₂-PEG-cRGD, and MnO₂-PEG-cRGD/Ce6 (n = 3). (F) UV-Vis spectra of Ce6, MnO2-PEG-cRGD, and MnO2PEG-cRGD/Ce6. Inset: UV-Vis spectra of MnO₂-PEG-cRGD nanosheets between 400 and 1000 nm.

Figure 2.

(A) Photothermal conversion curves of water and MnO₂-PEG-cRGD/Ce6 nanoparticles at different concentrations upon irradiation with an 660 nm laser (0.6 W cm^− 2). (B) Photothermal conversion curves of MnO₂-PEG-cRGD/Ce6 nanoparticles (100 µg mL^− 1) at different laser power densities. (C) Photothermal conversion stability of MnO₂-PEG-cRGD/Ce6 nanoparticles. The laser was turned on for 10 min and then turned off for each cycle. (D) Linear relationship between time and − ln θ calculated from cooling period after the laser was turned off.

Figure 3.

(A) TEM observation of MnO₂-PEG-cRGD/Ce6 after degradation in pH 5 containing 1 mM of H₂O₂ for 10, 30, and 60 min, respectively. (B) Generation of O₂ under the reduction of MnO₂-PEG-cRGD/Ce6 (50 µg mL^− 1) by 10 × 10^− 3 M H₂O₂ in acidic PBS (pH 5). (C) Consumption of DPBF over time due to ¹O₂ generation: (a) laser only, (b) MnO2-PEG-cRGD/Ce6 without laser, free Ce6, and (c) MnO2-PEG-cRGD/Ce6 with laser in the absence (d) or presence (e) of 100 mM H₂O₂ (pH 7.4). (D) Release profiles of Ce6 at different pHs with or without 660 nm NIR laser (0.6 W/cm2, 10 min).

Figure 4.

(A) Relative viability of PC3 and L929 cells after incubation with MnO2-PEG-cRGD (25, 50, 100, 200, 300, and 500 µg mL^− 1) for 24 h. (B) CLSM of intracellular ¹O₂ generation after PC3 cells were treated with PBS, MnO2-PEG-cRGD, free Ce6, and MnO2-PEG-cRGD/Ce6 under laser illumination. (C) CLSM of PC3 cells treated with MnO2-PEG/Ce6 and MnO2-PEG-cRGD/Ce6 (relative Ce6 = 5.0 μg/mL) for 4 h with or without laser illumination (Blue fluorescence is associated with DAPI; red fluorescence is expressed by released Ce6). Scale bars = 50 μm. (D) Bio-TEM images of PC3 cells incubated with MnO2-PEG/Ce6 and MnO2-PEGcRGD/Ce6 with or without laser irradiation. Scale bar: 1 μm.

Figure 5.

(A) Relative viabilities of PC3 cells after incubation with free Ce6, MnO2-PEG-cRGD with 660 nm light irradiation or MnO2-PEG-cRGD/Ce6 with or without 660 nm light irradiation (0.6 W cm^− 2, 10 min). ***p < .001, **p < .01. (B) Fluorescence images of calcein-AM (green)/PI (red) double stained cells after different treatments. (C) Flow cytometric analysis of cell apoptosis for 24 h caused by different treatments, using the Annexin V-FITC/PI staining. The four areas represent different phases of the cells: necrotic (Q1), late-stage apoptotic (Q2), early apoptotic (Q3), and live (Q4).