Polydopamine‐Modified Black Phosphorous Nanocapsule with Enhanced Stability and Photothermal Performance for Tumor Multimodal Treatments

Abstract As a novel 2D material, black phosphorus (BP) nanosheets are considered as a promising candidate for drug delivery platform for synergistic chemo/photothermal therapy. However, the intrinsic instability of bare BP poses a challenge in its biomedical applications. To date, some strategies have been employed to prevent BP from rapid ambient degradation. Unfortunately, most of these strategies are not suitable for the drug delivery systems. Here, a simple polydopamine modification method is developed to enhance the stability and photothermal performance of bare BP nanosheets. Then, this nanocapsule is used as a multifunctional codelivery system for the targeted chemo, gene, and photothermal therapy against multidrug‐resistant cancer. The enhanced tumor therapy effect is demonstrated by both in vitro and in vivo studies.


Materials
The bulk BP was purchased from Smart-Elements. Dopamine  Anti-Pgp antibody was purchased from Abcam (Cambridge, MA).

Preparation of BP nanosheets
The black phosphorus nanosheets were prepared using a simple modified liquid exfoliation of corresponding bulk sample. [1] In brief, 10 mg of bulk black phosphorus was dispersed in 20 mL of 1-Methyl-2-pyrrolidinone (NMP). The NMP was utilized to reduce the oxidation. The mixture solution was then sonicated in ice bath for 9 h with a sonic tip (Amplifier: 25%, On/Off cycle: 10 s/5 s). The ice water was used to avoid a relatively high temperature of the system. Afterward, the resulting brown suspension was centrifuged at 2000 rpm for 10 min to remove the residual unexfoliated bulk BP particles and the supernatant was carefully collected and stored under 4 °C for further use. Before use, the collected supernatant was centrifuged at 9000 rpm for 5 min to remove NMP.

Preparation of BP-siRNA
Briefly, 1 mg of BP was dispersed in 0.2 mL ethanol in 2 mL centrifuge tube, to which an aqueous solution of Guanidine Hydrochloride (4 M, 50 μL) and siRNA solution (1.5 nmol, dissolved in 50 μL DEPC-treated Milli-Q water) were added. The mixture was continuously shaken at 150 rpm for 0.5 h. The precipitate (BP-R) was collected by centrifugation at 9000 rpm for 5 min. The loading efficiency (LE) and encapsulation efficiency (EE) of siRNA were calculated using a previously published method. [2] Drug loading Chemotherapy DOX was loaded on BP NSs under a weakly alkaline condition in ethanol. In brief, 1 mg of BP-R NSs were mixed with 2 mL of DOX ethanol solution (1 mg mL -1 ), followed by an addition of 8 µL NaOH aqueous solution (20 mg mL -1 ).
After stirred in dark for 30 min, the obtained DOX loaded BP NSs (BP-R-D) were gathered by centrifugation at 9000 rpm for 5 min and washed with water. The DOX loading content (LC) was determined by a previously published method. [3]

Characterization of BP-based NSs
Transmission electron microscopy (TEM) images were acquired using FEI

Stability evaluation of BP and BP@PDA
To evaluate the influence of PDA encapsulation on BP stability, bare BP and BP@PDA NSs with the same amount of BP concentration (100 μg mL -1 ) were dispersed in water and exposed to air for 7 days and then their relevant properties were tested at predetermined time intervals.

Measurement of in vitro photothermal property
The h. After that, the cells were treated with NIR irradiation under 808 nm laser (1 Wcm -2 , 10 min). After incubation for an additional 12 h, the cell viabilities were evaluated by MTT assay. In brief, 10 μL MTT solution at a concentration of 5 mg mL -1 was added to the sample wells and incubated for a 4 h. Subsequently, the mixture was removed and 100 μL DMSO was added to the each well to dissolve the intracellular formazan crystals. The absorbance value was measured at 490 nm using a microplate reader (Bio-Rad Model 680, UK) after gentle shake for 10 min. The percentage of cell viability was measured relative to the media alone group (negative control).

In vitro Combined Antitumor Therapy
Wild-type and MDR MCF-7 cells were seeded on a 96-well plate at a density of where a and b represented length and width of the tumor, respectively.

Infrared Thermal Imaging
For the photothermal images, MCF-7/ADR tumor-bearing mice were intravenously injected separately with PBS (the control), BP@PDA-PEG and BP@PDA-PEG-Apt. After 24 h, the mice were anesthetized and the tumor sites were irradiated with a 808nm NIR laser at a power density of 1.5 W cm -2 for 5 min. During the irradiation, an infrared thermal image camera was used to monitor the temperature changes and infrared thermographic maps.

Biodistribution analysis
After treatments with DOX，BP-R-D@PDA-PEG and BP-R-D@PDA-PEG-Apt

Histological examination
For histological examination, organs including heart, liver, spleen, lung, and kidney were excised, fixed with 10% formalin, embedded with paraffin, sliced into thin sections and stained with hematoxylin and eosin (H&E).

Statistical analysis
Unless stated otherwise, all the experiments were carried out at least three times.
The experimental data are expressed as mean ± standard deviation (SD). Statistical analysis was performed by one-way ANOVA followed by Bonferroni test with SPSS 22.0 software. * P < 0.05 as statistical significance and ** P < 0.01 as extreme statistical significance.  Figure S1. The oxidative self-polymerization mechanism of dopamine and the conjugation mechanism between H 2 N-PEG-Apt and PDA coating.