Efficient Near Infrared Light Triggered Nitric Oxide Release Nanocomposites for Sensitizing Mild Photothermal Therapy

Abstract Mild photothermal therapy (PTT), as a new anticancer therapeutic strategy, faces big challenges of limited therapeutic accuracy and side‐effects due to uneven heat distribution. Here, near infrared triggered nitric oxide (NO) release nanocomposites based on bismuth sulfide (Bi2S3) nanoparticles and bis‐N‐nitroso compounds (BNN) are constructed for NO‐enhanced mild photothermal therapy. Upon 808 nm irradiation, the high photothermal conversion efficiency and on‐demand NO release are realized simultaneously. Due to the unique properties of NO, enhanced antitumor efficacy of mild PTT based on BNN‐Bi2S3 nanocomposites is achieved in vitro and in vivo. Mechanism studies reveal that the exogenous NO from BNN‐Bi2S3 could not only impair the autophagic self‐repairing ability of tumor cells in situ, but also diffuse to the surrounding cells to enhance the therapeutic effect. This work points out a strategy to overcome the difficulties in mild PTT, and has potentials for further exploitation of NO‐sensitized synergistic cancer therapy.


Experimental Section
Reagents: All chemicals were purchased from Sigma-Aldrich unless specified otherwise.

Calcein-AM (CA), propidium iodide (PI), Annexin V-FITC Apoptosis Detection Kit, and Cell
Counting  were provided by Dojindo Laboratories in Japan. DAF-FM and Hoechst 33342 were supplied from Beyotime Biotechnology. All the reagents were used without further purification. DI water was obtained by an 18.2 M recirculating deionized water system (arium pro DI, Sartorius).
Synthesis of Bi 2 S 3 nanorods: 2 mmol of Bi(NO 3 ) 3 was dispersed in 10 mL of OA and 10 mL of OM in a 100 mL flask. The solution was heated to 170 o C with stirring under Ar 2 atmosphere and kept at this temperature for 40 min. After the solution turned grey, a solution of 10 mmol of sulfur in 5 mL of OM was injected swiftly. The reaction was kept at 170 o C for 10 min and then stopped by injecting 40 mL of cold cyclohexane. The mixture was centrifuged at 8 000 rpm for 3 min, and the precipitate was collected and washed with hexane and ethanol (1:1 v/v) for 3 times.
Surface modification of Bi 2 S 3 using Tween-20: 100 L of Tween-20 was dispersed in 5 mL of cyclohexane and ultrasonic oscillated for 10 min. And then, 50 mg of the as-prepared Bi 2 S 3 nanorods was dispersed in 15 mL of cyclohexane, ultrasonic oscillated and injected into the previous solution. The mixture was heated to 70 o C to evaporate cyclohexane. When the cyclohexane was completely removed and the solution became oily, 40 mL of DI water was added and the mixture solution was ultrasonic oscillated. The mixed solution was sequentially kept at 70 o C to evaporate residual cyclohexane. The final product was obtained through centrifugation at 12 000 rpm for 10 min, and then washed with ethanol and water alternately for 3 times. NO donor: N, was synthesized as follows. 10 mmol of N,N'-bis-sec-butylamino-p-phenylenediamine (BPA) was diluted into 20 mL of ethanol, and then 20 mL of DI water containing 8.28 g of NaNO 2 was added under stirring and Ar 2 protection. After half hour, 20 mL of 6 M HCl was added slowly. Then, the mixture solution became yellow and precipitation appeared. After 6 h, the product was collected by centrifugation and washed with ethanol/water (1:1 v/v) and water in turn for 3 times. The solid product was freezing dried in dark condition.

Synthesis of
BNN loading and releasing: BNN was loaded in the Tween-20 layer on the surface of Bi 2 S 3 through an impregnation method. 20 mg of Tween-20 coated Bi 2 S 3 was dispersed in 20 mL of DI water, and then a solution of 20 mL of ethanol containing different amount of BNN was added. After stirring for 12 h in dark condition, the product was separated through centrifugation, and further washed by ethanol and water for several times to remove excess BNN. Meanwhile, the UV-Vis absorption spectra of the BNN supernatant collected after the first centrifugation procedure was measured. The loading percentage was calculated according to the standard curve of BNN in water/ethanol (1:1) solution. To test the stability, we investigated the release behavior of BNN from BNN-Bi 2 S 3 in PBS solution at 37 o C. Briefly, 10 mg of BNN-Bi 2 S 3 was dispersed in 20 mL of PBS solution containing 33 % ethanol, and then stirred at 37 o C. After stirring for the specific time (1h, 2h, 3h, 6h, 12h and 24h), 200 L of the solution was taken out. The samples were centrifugation at 12 000 rpm for 5 min, and the supernatant was collected to detect the concentration of BNN by UV-Vis absorption spectra. The final concentration was calculated according to the standard curve of BNN in PBS solusion (33% ethanol).
Calculation of the photothermal conversion efficiency: The energy conversion for the system can be expressed by equation 1 where m and C p are the mass and heat capacity, respectively. T refers to the solution temperature. Q NP represents the energy of nanoparticles, Q B is the baseline energy of the sample container, and Q sur is the heat conduction from the surface to air. Q NP could be calculated by equation 2.

808
(1 10 ) (2) where I is the laser power, is the photothermal conversion efficiency, and A 808 is the absorbance of BNN-Bi 2 S 3 at wavelength of 808 nm. Q sur is given by equation 3.
where h is heat transfer coefficient, S means the surface area of the sample container, and T r is the room temperature during the experiment. The heat input and output becomes balance when the system temperature reach a maximum. So, the photothermal conversion efficiency can be calculated by the following equation 4.
where Q B was calculated to be 58.2 mW in an independent experiment using the quartz cuvette filling with water. The (Tmax-Tr) was 20.3 o C according to Figure In cooling process, the laser is off, so Integral the equation 8, giving the expression So, time constant s is determined to be 344.4 as the slope from the cooling period versus negative natural logarithm of ( Supplementary Fig. S6b). Additionally, m is 1.0 g and C is 4.2 J g -1 . Thus, the photothermal conversion efficiency of BNN-Bi 2 S 3 can be calculated to be 33.8 %.
NO detection of BNN-Bi 2 S 3 under NIR irradiation: A fluorescent probe 2,3-Diaminonaphthalene (DAN, Sigma) was used to detect the release of NO from BNN-Bi 2 S 3 .
The standard curve should be established using NaNO 2 before the measurement. The as- Animal Technology Co. Ltd. All mice were acclimated to new environment for one week before treatment. When the tumor volume reached nearly 150 mm 3 , the mice were divided into 6 groups randomly (PBS, NIR, Bi 2 S 3 , BNN-Bi 2 S 3 , Bi 2 S 3 +NIR, BNN-Bi 2 S 3 +NIR). The intratumor injection dose of these nanoparticles were 20 mg kg -1 , and the NIR exposed time was 10 min (0.35 W cm -2 ). The temperature of the exposed area was recorded using an infrared thermal imager. The tumor size and mouse weight were recorded every two days.
The tumor sizes were measured by a caliper and calculated as volume = length x (width) 2 /2.
Finally, the mice were sacrificed, and tumors and major tissues were dissected to evaluate the therapeutic efficacy and bio-safety by H&E staining, Tunel staining and LC3 immunofluorescence assay. All procedures used were compliant with the Chinese Association for Laboratory Animal Sciences.
Detection of NO in tumor tissue: After different treatment, the tumor bearing mice were sacrificed and the subcutaneous tumors were collected. Then the tumors were lysed using the NO-specific lysis buffer (Beyotime Biotechnology) and tissue grinder. After that, the tissue lysates were collected to detect NO using the Nitric Oxide Assay Kit (Beyotime  Figure S1. a) SEM image of Bi 2 S 3 nanorods. b) EDX spectrum of Bi 2 S 3 nanorods.                 Figure S19. H&E stained tissue sections from the major organs (heart, liver, spleen, lung, and kidney) of mice after different treatment. The scale bar is 50 m.