Nanomedicine‐Enabled Photonic Thermogaseous Cancer Therapy

Abstract Local photothermal hyperthermia for tumor ablation and specific stimuliresponsive gas therapy feature the merits of remote operation, noninvasive intervention, and in situ tumor‐specific activation in cancer‐therapeutic biomedicine. Inspired by synergistic/sequential therapeutic modality, herein a novel therapeutic modality is reported based on the construction of two‐dimensional (2D) core/shell‐structured Nb2C–MSNs–SNO composite nanosheets for photonic thermogaseous therapy. A phototriggered thermogas‐generating nanoreactor is designed via mesoporous silica layer coating on the surface of Nb2C MXene nanosheets, where the mesopores provide the reservoirs for NO donor (S‐nitrosothiol (RSNO)), and the core of Nb2C produces heat shock upon second near‐infrared biowindow (NIR‐II) laser irradiation. The Nb2C–MSNs–SNO‐enabled photonic thermogaseous therapy undergoes a sequential process of phototriggered heat production from the core of Nb2C and thermotriggered NO generation, together with photoacoustic‐imaging (PAI) guidance and monitoring. The constructed Nb2C–MSNs–SNO nanoreactors exhibit high‐NIR‐induced photothermal effect, intense NIR‐controlled NO release, and desirable PAI performance. Based on these unique theranostic properties of Nb2C–MSNs–SNO nanocomposites, sequential photonic thermogaseous therapy with limited systematic toxicity on efficiently suppressing tumor growth is achieved by PAI‐guided NIR‐controlled NO release as well as heat generation. Such a thermogaseous approach representes a stimuli‐selective strategy for synergistic/sequential cancer treatment.


Measurement of NO Release.
The NO release from Nb 2 C-MSNs-SNO was qualitatively assessed and quantitatively measured using a typical Griess assay. To determine the cumulative release of NO from Nb 2 C-MSNs-SNO after different treatments, including different irradiation power densities (0, 0.5, 1.0, 1.5 and 2.0 cm -2 ) and varied concentrations ([Nb] = 0, 6.25, 12.5, 25, 50, 100 and 150 µg mL -1 ), the Griess agent was added into different groups and recorded by microplate reader. The fluorogenic probe, 3-Amino,4-aminomethyl-2',7'-difluorescein diacetate (DAF-FM DA), was applied to verify the intracellular NO release. 4T1 cells were pre-seeded into CLSM-specific dishes at a density of 1 × 10 5 and cultured for 12 h. After addition of Nb 2 C-MSNs-SNO at the concentration of 50 µg mL -1 and co-incubation for 12 h, the cells were incubated with DAF-FM DA (50 μM) for 20 min, and then exposed to 1064 nm laser at different power densities (0, 0.5, 1, 1.5, 2.0 W cm -2 ). The CLSM images of released NO were captured by excitation at 488 nm.

CLSM Analysis and Flow Cytometry Observation of Intracellular Endocytosis.
4T1 cells were seeded into CLSM-specific culture dishes (35 mm × 10 mm) and 6-well plates at a density of 1 × 10 5 and incubated for 24 h at 37 °C, following the medium was replaced by FITC-loaded Nb 2 C-MSNs-SNO (1 mL, [Nb] = 50 μg mL -1 ), which was then coincubated for 0, 1, 2, 4, and 8 h. Then, the medium was washed with PBS for 3 times, followed by cell nucleus was stained by DAPI for 20 min. CLSM imaging experiments were carried out on an Olympus FV1000 laser-scanning microscope equipped with a CW NIR laser (λ = 980 nm) as the excitation source. The flow cytometry was then used to evaluate intracellular endocytosis. Moreover, the mechanism of cellular uptake was investigated by pre-treatments of MβCD, sucrose, and amiloride for 30 min, followed by incubation with FITC-loaded Nb 2 C-MSNs-SNO (1 mL, [Nb] = 50 μg mL -1 ) for 4 h. To further quantify the intracellular fluorescence intensity, all cells were collected, and the fluorescence signals were measured.

In Vitro Synergistic therapy Effect of 4T1 Cells.
The 4T1 cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well for 12 h to attach on the plates, then coincubated with Nb 2 C-MSNs-PEG and Nb 2 C-MSNs-SNO at varied concentrations ([Nb] = 0, 12.5, 25, 50, 100, 200 μg mL -1 ) for 4 h. Following, these cells were exposed to 1064 nm laser irradiation for 5 min at 1.0 W cm -2 . In addition, to evaluate the cells viability after irradiation at different power densities (0, 0.5, 1.0, 1.5, and 2.0 W cm -2 ), 4T1 cells were coincubated with Nb 2 C-MSNs-PEG and Nb 2 C-MSNs-SNO at same concentrations for 5 min, then these cells were irradiated for 5 min using 1064 nm laser at different power densities. Finally, a standard CCK-8 protocol was used to assess cell viabilities.

Nb2C-MSNs-SNO.
Flow cytometry was applied to quantitatively assess cell apoptosis levels. Typically, 4T1 cells were seeded into 6-well plates for 24 h to attach on the dishes, then the medium was replaced by PBS, Nb 2 C-MSNs-PEG and Nb 2 C-MSNs-SNO ([Nb] = 100 μg mL -1 ) and incubated for 8 h. These cells were irradiated for 5 min using 1064 nm laser at different power densities (0, 0.5, 1.0 and 1.5 W cm -2 ). The cells were collected by dissociation via trypsin, centrifugation and washing for 3 times with PBS. Finally, the mixture solution, containing 5 μL PI and 5 μL FITC, was added into these cells for 20 min incubation. The flow cytometry was then used to evaluate cell-apoptosis levels. 4T1 cells were pre-seeded into CLSM-specific dishes at a density of 1 × 10 5 and cultured for 12 h to attach on the plates. Then, the PBS, Nb 2 C-MSNs-PEG and Nb 2 C-MSNs-SNO ([Nb] = 100 μg mL -1 ) were co-incubated with 4T1 cells for 4 h. Then, these cells were irradiated for 5 min using 1064 nm laser at different power densities (0, 0.5, 1.0 and 1.5 W cm -2 ). After treatments, 4T1 cells were observed by CLSM, in which live cells and dead cells were stained by Calcein-AM and PI, respectively.

In Vivo Toxicity Assay.
Animal experiment procedures were confirmed to the guidelines for the Animal Care

In Vivo Blood Circulation, Biodistribution and Metabolism of Nb 2 C-MSNs-SNO.
For pharmacokinetic analysis, five healthy female Kunming mice were intravenously administered with Nb 2 C-MSNs-SNO, followed by 10 μl blood samples were collected and immersed into saline (1 mL) containing heparin sodium (50 unit mL -1 ) at varied time points (1, 5, 10, 15, and 30 min, 1, 2, 4, 8, 10, and 24 h). The Nb amounts of these samples were determined by ICP-OES. For biodistribution analysis, 4T1 tumor-bearing mice were randomly divided into 6 groups (n = 3) when the tumor volume reached around 150 ~ 200 mm 3 and then intravenously administered with Nb 2 C-MSNs-SNO. Then, the tumors and major organs (heart, liver, spleen, lung and kidney) were collected at different time points (1, 2, 4, 8, 12, and 24 h) of post-injection, following these tumor and organs were weighed and homogenized. ICP-OES was used to determine the Nb amount in these organs/tissues. The metabolism process of Nb 2 C-MSNs-SNO was assessed in female Kunming mice (n = 4), Nb 2 C-MSNs-SNO in PBS ([Nb] = 5 mg kg -1 ) was intravenously administered into the mice.
The faeces and urine were collected at varied time points (2, 6, 12, 24, 36, and 48 h) and the Nb content in faeces and urine was determined by ICP-OES.

In Vivo Photonic Thermogaseous Cancer Therapy against Tumor-Bearing Mice.
To establish xenograft tumor model, 4T1 cells (1 × 10 6 cell/site) dispersed in saline solution (100 μL for each mouse) were injected into the back of mice, which were the healthy female Balb/c nude mice (5 weeks old). These mice were randomly divided into six groups (n = 5), including (1)  determined in 14-day period after synergistic therapy. Subsequently, the tumors and major organs (heart, liver, spleen, lung and kidney) were sliced and stained with H&E, TUNEL and Ki-67 for histological analysis. Furthermore, to understand the mechanism of tumor growth inhibition, the apoptotic proteins (Bid, Caspase-3, Caspase-7) expression levels were tested.