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

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 αvβ3 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 αvβ3, 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 αvβ3 overexpressed.

Optoelectronics Technology Co. Ltd., Shanghai, China). The temperature was recorded every 5 s by an online DT-8891 Ethermocouple thermometer (Shenzhen Everbest Machinery Industry Co., Ltd., Shenzhen, China). In order to investigate the photothermal stability, five cycles of repeated laser irradiation were applied to ICG, LAP, ICG/LAP, LAP-PDA and ICG/LAP-PDA solutions. For each cycle, the solution was heated by an 808 nm laser irradiation (0.25 cm 2 , 1.2 W/cm 2 , 3 min) and followed by a cooling period to room temperature. The solution temperature was recorded every 5 s by thermometer.

Cell Culture and Cytotoxicity Assay
Human breast cancer MDA-MB-231 cells overexpressing integrin αvβ3 were cultured and passaged in 25-cm 2 plates with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin under 37 °C and 5% CO2.
CCK-8 assay was used to quantify the viability of MDA-MB-231 cells after treated with LAP-PDA-PEG-RGD, ICG/LAP-PDA-mPEG or ILPR NPs at different ICG concentrations. Briefly, MDA-MB-231 cells were seeded in 96-well plates at a density of 1×10 4 cells/well with 100 μL of fresh DMEM at the day before the experiment. After 24 h, the medium was replaced by 100 μL fresh medium containing PBS (control), LAP-PDA-PEG-RGD, ICG/LAP-PDA-mPEG and ILPR NPs with different ICG concentrations (5, 10, 20, 30 and 40 μg/mL). After 24 h incubation, the medium was discarded and the cells were washed with PBS for 3 times, followed by addition of 100 μL fresh DMEM containing 10 μL CCK-8. After incubation of the cells at 37 °C for another 3 h, the absorbance were measured by Thermo Scientific Multiskan MK3 ELISA reader (Thermo Scientific, Hudson, NH) at 450 nm in each well. The standard deviation of 5 wells of each sample was recorded.

In Vitro Cellular Uptake Assay
MDA-MB-231 cells were seeded into 12-well plates at a density of 2×10 5 cells/well at 37 °C and in 5% CO2 atmosphere overnight, and then the medium was replaced with fresh medium containing ICG/LAP-PDA-mPEG and ILPR NPs at different ICG concentrations (5, 10, 20, 30 and 40 μg/mL). After 6 h incubation, the medium was removed carefully and the cells were washed with PBS for 3 times, trypsinized, centrifuged, and counted by Handheld Automated Cell Counter (Millipore, Billerica, MA). The remaining cells were lysed using aquaregia solution (1.0 mL) for 2 days, and then the Si concentration in the cells was quantified by ICP-OES to investigate the cellular uptake of ICG/LAP-PDA-mPEG and ILPR.

ROS Production
1,4-diphenyl-2,3-benzofuran (DPBF) was used as a ROS chemical probe to evaluate the 1 O2 generation capability of ILPR. Specifically, 2 mL of a DPBF/DMF solution (20 μM) was added to 2 mL of water, LAP, ICG, LAP-PDA-PEG-RGD and ILPR aqueous solution, respectively. The mixture was irradiated by an 808 nm laser (1.2 W/cm 2 ) for 8 min. Then the absorption intensity of DPBF in different solutions at 417 nm was evaluated by UV-vis spectroscopy. To evaluate the ROS production in cells, MDA-MB-231 cells were seeded into 12-well plates at a density of 2×10 5 cells/well at 37 °C and in 5% CO2 atmosphere overnight, and then the medium was replaced with fresh medium containing ILPR or LAP-PDA-PEG-RGD at the same ICG concentrations (10, 40 μg/mL). After 6 h incubation, the medium was removed carefully and the cells were washed with PBS for 3 times. Then the cells were irradiated under an 808 nm laser (1.2 W/cm 2 , 5 min). Furthermore, after NIR laser irradiation, 100 μL medium containing DCFH-DA (25 mM) was added in each well for 45 min and then the medium was replaced with PBS.
[40] The fluorescence signal in cells was observed by a fluorescence microscopy (CARL Zeiss, Axio Vert. A1, Germany), and the fluorescent intensity was detected using a FACScan Calibur flow cytometer (Becton Dickinson, Mountain View, CA) [41].

PTT and PDT of Cancer Cells in Vitro
To estimate the influence of laser power density on the PTT and PDT therapeutic effect of different materials, MDA-MB-231 cells were incubated with LAP-PDA-PEG-RGD, ICG/LAP-PDA-mPEG and ILPR at the same ICG concentration of 40 μg/mL for 6 h, and then irradiated under an 808 nm laser (2.5 cm 2 , 5 min) at different power densities (0.8, 1.0, 1.2 W/cm 2 ). Then, MDA-MB-231 cells were incubated with different materials at different ICG concentrations (10, 20 and 40 μg/mL) for 6 h, and then irradiated under an 808 nm laser (2.5 cm 2 , 1.2 W/cm 2 , 5 min). The cell viability was evaluated by CCK-8 assay performed by previous protocol and the standard deviation of 5 wells of each sample was recorded.
MDA-MB-231 cells were incubated with LAP-PDA-PEG-RGD, ICG/LAP-PDA-mPEG and ILPR at the same ICG concentration of 40 μg/mL for 6 h, and then irradiated under an 808 nm laser (1.2 W/cm 2 , 0.25 cm 2 , 5 min). Then the cells were stained by PI (8 μM) and Calcein-AM solution (2 μM) for 45 min at room temperature, and rinsed with PBS for 3 times. Finally, the cells were observed via a fluorescence microscopy (CARL Zeiss, Axio Vert. A1, Germany).

Statistical Analysis
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 S1. Photographs of ICG, LAP and ICG/LAP before centrifugation (left) or after centrifugation (right).

Before centrifugation
After centrifugation S4   Table S1. Optimization of ratio for ICG loading (the LAP concentration and ICG concentration were fixed at 2 mg/mL).       LAP-PDA-PEG-RGD were synthesized as a control material without loading ICG and evaluated by DLS (Table S2) and 1H NMR (Fig. S8). After the modification of mPEG and PEG-RGD, the hydrodynamic size of NPs slightly increased from 89.2 nm to 104.2 nm and 109.6 nm, indicating that formed nanoparticles still possess favourable size and good distribution for further biomedical application. Meanwhile, the surface potential increased from -32.3 mV of LAP-PDA to -29.5 mV of LAP-PDA-mPEG and -27.8 mV of LAP-PDA-PEG-RGD, suggesting the successful modification of PEG chains. The formed LAP-PDA-mPEG and LAP-PDA-PEG-RGD were characterized by 1H NMR spectroscopy (Fig. S8). Since the chemical shift of PEG chains at 3.7 ppm overlapped with that of PDA, an enhanced peak at 3.7 ppm could be observed in the 1H NMR spectrum of LAP-PDA-mPEG. After the modification of RGD-PEG, the RGD-associated aromatic proton peaks at 7.3 and 7.4 ppm appeared in the 1H NMR result of LAP-PDA-PEG-RGD, indicating that LAP-PDA-PEG-RGD was synthesized successfully.