Near-Infrared Light-Triggered Sulfur Dioxide Gas Therapy of Cancer

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Near-Infrared Light Triggered Sulfur Dioxide Gas Therapy of Cancer Shihua Li, Rui Liu, Xiaoxue Jiang, Yuan Qiu, Xiaorong Song, Guoming Huang, Nanyan Fu, Lisen Lin, Jibin Song, Xiaoyuan Chen, and Huanghao Yang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b08700 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 15, 2019

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Near-Infrared Light Triggered Sulfur Dioxide Gas Therapy of Cancer

Shihua Li,† Rui Liu,‡ Xiaoxue Jiang,† Yuan Qiu,‡ Xiaorong Song,† Guoming Huang,‡ Nanyan Fu,† Lisen Lin, § Jibin Song,*,† Xiaoyuan Chen,*,§ and Huanghao Yang*,†

†

MOE Key Laboratory for Analytical Science of Food Safety and Biology, State Key

Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350116, P. R. China. ‡

College of Biological Science and Engineering, Fuzhou University, Fuzhou 350116,

P. R. China. §

National Institute of Neurological Disorders and Stroke (NINDS), National Institutes

of Health (NIH), Bethesda, Maryland 20892, United States

ABSTRACT The exploitation of gas therapy platforms holds great promise as a "green" approach for selective cancer therapy, however, it is often associated with some challenges, such as uncontrolled or insufficient gas generation and unclear therapeutic

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mechanisms. In this work, a gas therapy approach based on near-infrared (NIR) light triggered sulfur dioxide (SO2) generation was developed, and the therapeutic mechanism as well as in vivo anti-tumor therapeutic efficacy was demonstrated. A SO2 prodrug-loaded rattle-structured upconversion@silica nanoparticles (RUCSNs) was constructed to enable high loading capacity without obvious leakage, and to convert NIR light into ultraviolet (UV) light so as to activate the prodrug for SO2 generation. In addition, SO2 prodrug-loaded RUCSNs showed high cell uptake, good biocompatibility, intracellular tracking ability, and high NIR light triggered cytotoxicity. Furthermore, the cytotoxic SO2 was found to induce cell apoptosis accompanied with the increase of intracellular reactive oxygen species (ROS) levels and the damage of nuclear DNA. Moreover, efficient inhibition of tumor growth was achieved, associated with significantly prolonged survival of mice. Such NIR light-triggered SO2 therapy may provide an effective strategy to stimulate further development of synergistic cancer therapy platforms.

KEYWORDS: gas therapy, sulfur dioxide (SO2), upconversion nanoparticles (UCNPs), near-infrared (NIR), cancer therapy

Gas therapy based on gaseous molecules is a promising treatment method for various diseases, ranging from wound, inflammatory and cardiovascular diseases to cancers.1-4 More specifically, in gas therapy of tumors, gaseous molecules such as


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nitric oxide (NO),5-8 hydrogen sulfide (H2S),9,

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carbon monoxide (CO),11-13 carbon

dioxide (CO2),14 and hydrogen (H2)15, 16 at elevated concentrations can kill cancer cells. Interestingly, these gaseous molecules, which are important physiological messenger molecules, and necessary for maintaining the homeostasis of physiological functions in the body,17-19 thus exerting negligible side effects on normal cells/tissues.2 It has been recently found that gas therapy can be combined with other treatments for synergistic cancer therapy.20-22 For example, NO molecules can enhance the chemosensitivity of cancer cells through the reduction of P-glycoprotein expression, overcoming multidrug resistance, 8, 21, 22 and CO molecules can sensitize cancer cells to chemotherapy by enhancing mitochondrial biogenesis and aggravating the oxidative stress of cancer cells.13 Therefore, the development of high-efficiency gas therapy paradigms is of high significance and demand. As a member of the gasotransmitter family, sulfur dioxide (SO2) has been traditionally known as an air pollutant. At elevated concentrations, SO2 has a toxicological effect by inhalation, which might contribute to oxidative stress-induced damage of biomacromolecules such as proteins, lipids, and DNAs.23-25 In recent reports, intriguing roles of SO2 in fighting diseases have been revealed, including its use as a vasorelaxant substance,26 as an antimycobacterial agent,27 and as a synergistic treatment to overcome drug-resistance in cancer chemotherapy.25 However, currently it still lacks the deeper understanding of in vitro and in vivo therapeutic effect of SO2 for cancer therapy. Besides, the low stability and biocompatibility, and the limited SO2 accumulation in specific tissues of current donors


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limits the in vivo therapeutic applications of SO2 gas.28,

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In this context, the

development of a therapeutic system based on the effective delivery and on-demand generation of SO2 may provide a good platform for effective cancer therapy with negligible side effects, which yet remains elusive.

Figure 1. (A) Schematic illustration of the preparation of RUCSNs-DM with DM loading in the pores and the interior cavity of the RUCSNs, and SO2 generation upon NIR light irradiation. (B) Intracellular localized SO2 generation and therapeutic action upon NIR light irradiation after cell uptake of RUCSNs-DM.

Herein, a platform for cancer therapy by remote-controlled supply of SO2 based on


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prodrug-loaded rattle-structured upconversion@porous silica nanoparticles (RUCSNs) was designed (Figure 1). The SO2 prodrug selected in this work is 1-(2,5dimethylthien-1,1-dioxide-3-yl)-2-(2,5-dimethylthien-3-yl)-hexafluorocyclopentene (DM) molecule. The DM-loaded RUCSNs was named RUCSNs-DM. RUCSNs not only enable high SO2 prodrug loading capacity, but also convert near-infrared (NIR) photons to high-energy photons,30-33 which stimulate SO2 release through the photolysis of the prodrug. The developed therapeutic system was not only able to effectively solve the intractable problems associated with biocompatibility, and intracellular and in vivo delivery of the SO2 prodrug, but also achieved on-demand release of SO2 in deep tissues using NIR light and reduced the phototoxicity caused by ultraviolet (UV) light excitation. Upon NIR light irradiation, the intracellular generation of SO2 was able to modulate oxidative stress by increasing the reactive oxygen species (ROS) levels and further cause DNA damage, which finally resulted in cancer cell death through apoptosis. As an efficient form in gas therapy, the investigated NIR-responsive SO2 platform can be a promising alternative for boosting the development of anti-tumor gas therapies.

RESULTS AND DISCUSSION Synthesis and characterizations of RUCSNs.


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Figure 2. Synthesis and characterizations of RUCSNs. (A) TEM image of NaYF4: Tm/Yb@NaYF4. Inset: the particle size distribution of NaYF4: Tm/Yb@NaYF4. (B) Emission spectra of core UCNPs, core-shell UCNPs and RUCSNs exposed to 980 nm laser (1 W/cm2). (C) Schematic illustration of energy level transfer from Yb3+ to Tm3+ in NaYF4: Tm/Yb. (D) Luminescence images of the solution of the core UCNPs, coreshell UCNPs and RUCSNs at 500 μg/mL with the irradiation of 980 nm laser (1 W/cm2). (E) TEM image of RUCSNs after the hot water etching. (F) The N2 adsorptiondesorption isotherms and pore size distribution of RUCSNs. (G) Elemental mapping of RUCSNs including Y, Yb, Tm, and Si elements.

In general, the SO2 prodrug in this study needs to be activated by UV light, which greatly hampers its further in vivo application due to the low tissue penetration depth


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and the high phototoxicity of UV light.34 Thus, upconversion nanoparticles (UCNPs) were chosen as photo-regulators, which can convert NIR light to UV light, allowing for increased tissue penetration depth and lower phototoxicity.35-37 Initially, core-shell UCNPs (NaYF4: 0.3% Tm / 25% Yb @NaYF4) were synthesized through seedmediated epitaxial shell growth by a high temperature co-precipitation method.38, 39 Transmission electron micrographs (TEM) showed an increase in particle size from ~27.0 nm (Figure S1) to ~32.2 nm (Figure 2A) after shell growth, indicating that the shell thickness was approximately 5.2 nm. High resolution TEM (HRTEM) analysis of the as-prepared core UCNPs (NaYF4: Tm/Yb) revealed a d-spacing of 0.29 nm (Figure S2), corresponding to the lattice spacing in the (110) planes of hexagonal NaYF4. Good crystallization of the UCNPs was also confirmed by X-ray diffraction (XRD) patterns (Figure S3). Upon the excitation of 980 nm laser, core UCNPs displayed intense upconversion luminescence with a set of sharp emissions centered at 347, 362, 450, 483, and 648 nm (Figure 2B), which were assigned to the 1I6 → 3F4, 1D2 → 3H

6,

1D

2

→ 3F4, 1G4 → 3H6, and 1G4 → 3F4 transitions of Tm3+, respectively (Figure 2C).

In addition, luminescence intensities of RUCSNs were examined by using different irradiation power, which showed power-dependent enhancement of luminescence (Figure S4). In order to avert the possible skin injury under high laser power, we chose irradiation laser power of 1 W/cm2 in our experiments. More importantly, core-shell structured UCNPs showed ~20-fold enhancement of luminescence compared with that of core UCNPs, due to the increased surface quenching effects in the core-shell structure (Figure 2B). Besides, the luminescence brightness of the core-shell


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structured UCNPs solution was much higher than that of the core UCNPs under the irradiation of 980 nm laser (Figure 2D). In order to endow the UCNPs with cargo loading capability, an amorphous silica shell was grown on the UCNPs surface using a reverse microemulsion method (Figure S5).40,

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Subsequently, a hot water etching approach was employed to render the

formation of RUCSNs (composed of NaYF4:Yb/Tm@NaYF4@SiO2) by heating the solution to 96 °C and etching for 6 h under the protection of polyvinylpyrrolidone (PVP) which can as a surface protecting agent to avoid the aggregation of nanoparticles due to the robust hydrogen bonds between carbonyl groups in PVP and hydroxyls in silica.40-42 The resulted rattle-structure had an overall size of around 65 nm and an outer mesoporous silica shell of around 10 nm (Figure 2E). In addition, the dynamic light scattering (DLS) of RUCSNs before and after etching were examined (Figure S6), which showed a slightly increased hydrodynamic diameter after etching, which is possibly due to the surface PVP coating. As shown in Figure 2F, N2 adsorptiondesorption analysis and pore-size distribution verified the mesoporous structure of the RUCSNs, with a Brunauer-Emmett-Teller (BET) surface area of 411 m2/g and pore size of about 2.5 nm. Moreover, a typical strong upconversion emission of Tm3+ was observed in the RUCSNs, indicating the preservation of the optical properties of the inner UCNPs after surface silica coating (Figure 2B and 2D). Elemental mapping clearly revealed that the Tm/Yb ions were successfully doped in NaYF4 and the outer silica was coated on the UCNPs (Figure 2G). According to the above characterizations, RUCSNs with good morphology, large surface area, and high NIR light excited UV


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emission were constructed, providing the preconditions for prodrug loading and NIR light-triggered SO2 generation.

NIR light-triggered SO2 gas generation

Figure 3. Characterization of NIR light-triggered SO2 generation of RUCSNs-DM. (A) Schematic illustration of NIR light-triggered SO2 generation from RUCSNs-DM. (B) UV-vis absorption spectra of DM before and after UV irradiation (365 nm). (C) UV-vis absorption spectra of RUCSNs, RUCSNs-DM, and RUCSNs-DM without or with NIR light treatments. (D) Emission spectra of RUCSNs and RUCSNs-DM exposed to 980 nm laser. (E) Fluorescence intensities of the DEACA probe after incubation with RUCSNs, DM and RUCSNs-DM under irradiation with 980 nm laser for different periods of time. Data points represent mean ± s.d. (n = 3).


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To allow photo-controlled generation of SO2, a diarylethene derivative DM was chosen as the SO2 prodrug, which has been reported to be an efficient SO2 generator upon UV light exposure.29 Considering that organic prodrug molecules are chemically unstable and have low tumor accumulation,43 in this study, DM was loaded into the nanoplatform in order to enhance biocompatibility and conquer toxic side effects of the prodrugs. The DM was able to release SO2 by C-S bond cleavage or molecular dimerization under UV light irradiation (Figure 3A). Nuclear magnetic resonance (NMR) results demonstrated successful synthesis of DM (Figure S7 – S10). The absorption spectrum showed broad absorption bands between 200 - 400 nm (Figure 3B). Furthermore, UCNPs were chosen to convert the NIR light into UV light, in order to excite the DM molecules. Benefiting from the increased photo-stability of the UCNPs and the high tissue-penetration depth of NIR light,44-47 the combination of UCNPs with DM was expected to achieve high DM excitation efficiency for robust tumor therapy in

vivo. In the next step, DM was encapsulated in the pores and the interior cavity of the RUCSNs, resulting in the formation of RUCSNs-DM (Figure S11). Due to the partial hydrophobicity of DM and the electrostatic force between the negatively charged SiOH group and the F electron-drawing groups of DM, DM was able to be strongly encapsulated inside the RUCSNs. RUCSNs-DM showed increased absorption compared to the simple RUCSNs (Figure 3C), which was caused by the loaded DM in the nanoplatform. By calculating the load capacity through absorption spectra, RUCSNs-DM exhibited a high loading content of ~9.5 wt% (Figure S12). RUCSNs-


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DM was dispersed in water, phosphate buffered saline (PBS), fetal bovine serum (FBS), or cell culture medium to test its dispersion stability. These RUCSNs-DM aqueous dispersions showed no aggregation or precipitation even after 12 days of incubation. DLS data also revealed good dispersibility of RUCSNs-DM with negligible size variation after incubation with various media (Figure S13). Furthermore, the leakage experiment of RUCSNs-DM revealed only 7.4 % and 12.6% DM leakage after being dispersed in PBS at pH 7.4 and pH 5.0 for 48 h, respectively (Figure S14), proving excellent DM loading stability in the RUCSNs. In the luminescence study under 980 nm laser excitation, RUCSNs-DM exhibited an obvious decrease (~70%) in UV emissions (347 nm, 362 nm) as well as unchanged blue (450 nm, 483 nm) and red emissions (648 nm) as compared to those of the RUCSNs, suggesting efficient energy transfer of UV emissions to DM (Figure 3D). To study the photoinduced release of SO2 from RUCSNs-DM, the SO2 generation was monitored with a 7-diethylaminocoumarin-3-aldehyde (DEACA) probe, which was able to react with bisulfite anion through a nucleophilic addition reaction to significantly enhance the luminescence.48 After irradiating the RUCSNs-DM with a 980 nm laser, the probe displayed increased blue fluorescence at 483 nm with prolonged irradiation time (Figure 3E), suggesting NIR light-triggered SO2 release. In contrast, the probe fluorescence was not significantly enhanced in isolated RUCSNs or DM irradiation at 980 nm, indicating that SO2 was controllably produced by applying 980 nm laser stimuli. Therefore, the release rate of SO2 gas was tuned by changing the laser irradiation time and power, which can further modulate the gas treatment efficacy.


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RUCSNs-DM in vitro therapy studies upon NIR light irradiation

Figure 4. In vitro therapeutic study of RUCSNs-DM in cancer cells. (A) Relative cell viabilities of HeLa, MCF-7, and S180 cells treated with different concentrations of RUCSNs for 24 h. (B) Relative cell viabilities of HeLa cells treated with different concentrations of RUCSNs-DM without or with 980 nm laser irradiation (1 W/cm2, 10 min). (C) Fluorescence images of HeLa cells co-stained by Calcein-AM (green fluorescence, live cells) and PI (red fluorescence, dead cells) after treatment with PBS (control), RUCSNs, RUCSNs-DM, and RUCSNs-DM with 980 nm laser irradiation (1 W/cm2, 10 min). Data points represent mean ± s.d. (n = 3). Scale bar: 100 μm.

Before evaluating the NIR light-triggered therapeutic performance of RUCSNs-DM, its cytotoxicity and treatment feasibility were surveyed. According to the CCK-8 assay


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results, all of the HeLa, MCF-7, and S180 cells maintained high cell viability of > 90% after 24 h incubation with RUCSNs, even when the concentration reached 500 µg/mL (Figure 4A), suggesting low RUCSNs cytotoxicity. More significantly, after DM loading in the RUCSNs, RUCSNs-DM exerted negligible cytotoxicity on cancer cells without NIR light irradiation, due to the biocompatibility of silica shell and its increased stability without DM leakage (Figure 4B). Subsequently, the NIR light-triggered therapeutic effect of RUCSNs-DM on cancer cells was evaluated. After treating HeLa cells with various concentrations of RUCSNs-DM, cell viability showed a concentrationdependent decrease after 980 nm laser irradiation (Figure 4B). After using MCF-7 and S180 cells other cancer cell models, similar high therapeutic efficacy was achieved (Figure S15 and Figure S16). The cell uptake efficacy was investigated after treating HeLa cells with RUCSNs-DM for 16 h. Thin-section cell TEM analysis revealed efficient accumulation of RUCSNs-DM in the cells, indicative of its high cell uptake (Figure S17). In order to gain more insight, live/dead cell double-staining experiments were performed by using Calcein-AM and propidium iodide (PI), which showed green and red fluorescence in the confocal imaging system, respectively (Figure 4C and Figure S18). It could be easily identified that the lethal rate of HeLa cells with RUCSNsDM treatment plus 980 nm laser irradiation was much higher than those in the other three groups (control group, RUCSNs group, RUCSNs with NIR group, and RUCSNsDM without NIR group). All the above-mentioned cytotoxicity evaluations validated that RUCSNs-DM can act as a NIR-controllable gas therapy platform for killing cancer cells.


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SO2 generation and therapeutic action in cancer cells

Figure 5. (A) Confocal imaging of intracellular ROS levels in HeLa cells after treatment with PBS (control), RUCSNs, RUCSNs with 980 nm laser irradiation, RUCSNs-DM, and RUCSNs-DM with 980 nm laser irradiation (1 W/cm2, 10 min). Scale bar: 100 μm. (B) Intracellular TUNEL staining in HeLa cells after treatment with different formulations (blue fluorescence: DAPI, green fluorescence: TUNEL). The yellow arrows indicated the overlap of blue fluorescence from DAPI and the green fluorescence from TUNEL, suggesting the DNA fragmentation in nucleus. Scale bar: 50 μm.

To study the SO2 gas cancer therapy mechanism, in vitro experiments were performed. Firstly, intracellular delivery of RUCSNs-DM was studied by confocal laser


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scanning microscopy (CLSM). After incubating cancer cells with RUCSNs-DM for 2 h, the green fluorescence of UCNPs overlapped with the red fluorescence of lysosomes stained by LysoTracker Red, which led to yellow spots in the merged image (Figure S19). In addition, the Pearson's correlation coefficient was calculated to study the colocalization of UCNPs and LysoTracker channels, which showed a weaker correlation at the 5 h post incubation group compared to that at 2 h post incubation group (Figure S19), suggesting the possible lysosome escape of RUCSNs-DM. In the next step, the intracellular oxidative stress after RUCSNs-DM treatment and NIR irradiation was investigated. A 2,7-dichlorofuorescin diacetate (DCFH-DA) probe was used to monitor the intracellular ROS level, which can be hydrolyzed by intracellular esterase to produce non-fluorescent DCFH and can be further converted to fluorescent dichlorofluorescein (DCF) after oxidization by intracellular ROS. The results showed that cells treated with RUCSNs-DM and NIR light displayed prominent green fluorescence, while no obvious fluorescence could be observed in the other four groups (Figure 5A), indicating elevated intracellular ROS level during NIR irradiation of RUCSNs-DM. Furthermore, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was carried out to identify the cell death pathway.49 In the TUNEL experiment, once nucleus DNA was broken at the late stage of apoptosis, cells would produce a large number of cohesive 3'-OH terminals, which could bind with the fluorescein- or enzyme-labeled dUTP under terminal deoxynucleotidyl transferase (TdT) catalysis. Thus, the occurrence of apoptosis was identified by visualizing the green fluorescence of fluorescein. 4’,6-diamidino-2-phenylindole (DAPI) staining with


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blue fluorescence was used to label the nucleus. After treating the cells with RUCSNsDM and NIR irradiation, cell nuclei displayed varying degrees of green fluorescence, which suggested that the cells underwent apoptosis and DNA fragmentation in nucleus (Figure 5B), confirming that NIR light-triggered intracellular SO2 generation from RUCSNs-DM greatly increased the concentration of ROS and led to cell apoptosis with DNA damage. Currently, the precise mechanisms of SO2 therapy are still not fully understood. Based on our findings, we found the effective therapy of SO2 and identified the DNA damage and elevated ROS levels upon SO2 generation. Besides, previous works have revealed the ability of SO2 in tuning ion channels and stimulating cAMP/PKA signal pathway,50 which might also result in the inhibited tumor cell proliferation.


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Figure 6. Cell apoptosis studies with the staining of annexin V-FITC and PI dyes in HeLa cells. (A) Flow cytometry assays of HeLa cells treated with PBS (control), RUCSNs, RUCSNs with NIR, RUCSNs-DM, RUCSNs-DM with NIR. (B) CLSM images of HeLa cells stained with annexin V-FITC/ PI after the treatment of RUCSNs-DM with NIR light irradiation for 0, 3, 6, 10, and 15 h. Scale bar: 20 μm.

Annexin V, a phospholipid-binding protein, has high affinity for phosphatidylserine that can invert to the outer side of cell membrane during cell apoptosis. PI is a nucleic acid dye, which cannot permeate the integrated cell membrane of live or early apoptotic cells, but can pass through the disruptive cell membrane and nucleus of late


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apoptotic or dead cells. To further confirm apoptosis, flow cytometry experiments and Annexin V-FITC/PI co-staining assay were carried out. In flow cytometry analysis (Figure 6A), an increased proportion of apoptotic cells (48.5%) and a reduced proportion of live cells (48.7%) were observed in the group treated with RUCSNs-DM and NIR irradiation compared to those of the other three groups, indicating activation of apoptotic pathways. Furthermore, CLSM indicated the different stages of apoptosis in HeLa cells from the early to the late apoptotic stage (Figure 6B and Figure S20). In detail, HeLa cells were treated with RUCSNs-DM for different times (0, 3, 6, 10, and 15 h) after 980 nm laser irradiation, and then were stained with Annexin V-FITC (green fluorescence) and PI (red fluorescence). After incubation for 3 h to 10 h, most of the cells were stained only with Annexin V-FITC, indicating that the cells were in the early apoptotic stage. However, after extending the treatment time, most cells moved to the late apoptotic stage (both green and red fluorescence). Taken together, it was confirmed that RUCSNs-DM with efficient SO2 production may serve as a cancer gas therapy platform based on controllable NIR-activated apoptosis.

In vivo biosafety and biodistribution of RUCSNs-DM


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Figure 7. Biosafety assays and biodistribution of RUCSNs-DM. (A) Hemolysis assays of RUCSNs-DM with different concentrations. Water and PBS were acted as positive and negative control respectively. (B) Blood biochemistry assays of liver function markers: ALT, AST, and ALP. (C) Blood biochemistry assays of kidney function markers: BUN and CRE. (D) Biodistribution of RUCSNs-DM in major organs and tumor tissues at 24 h and 14 d post intravenous injection of RUCSNs-DM (20 mg/kg). Data points represent mean ± s.d. (n = 3).

In order to evaluate the biosafety of RUCSNs-DM, hematology-related assays were carried out prior to in vivo therapeutic experiments. Hemolysis against red blood cells was employed to evaluate the blood-contact safety of RUCSNs-DM. Negligible


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hemolytic activity (percent hemolysis < 4%) at 1000 μg/mL RUCSNs-DM concentration was shown, which indicated good hemocompatibility (Figure 7A). Blood biochemistry assays were conducted, including analysis of liver function markers (alanine

transaminase

(ALT),

aspartate

aminotransferase

(AST),

alkaline

phosphatase (ALP)) and kidney function markers (blood urea nitrogen (BUN) and creatinine (CRE)). No visible hepatic or renal toxicity was observed at 24 h, 14 d, and 30 d post intravenous injection of RUCSNs-DM (Figure 7B and 7C). In addition, standard hematological biomarkers, including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), and platelets (PLT) were chosen for blood chemistry analysis (Figure S21). All these blood parameters showed no distinct abnormality before and after injection for 24 h, 14 d, and 30 d, indicating negligible side effects of RUCSNs-DM on the hematological system. All these hematology analyses showed the excellent biosafety of RUCSNs-DM. In order to investigate the in vivo biodistribution of RUCSNs-DM, tumor-bearing Balb/c mice were firstly prepared by hypodermic inoculation of S180 cells (1×106) in the right hind leg of mice. When the tumor size reached ~50 mm3, the major organs of the tumor-bearing Balb/c mice, including heart, liver, spleen, lung, kidney, and tumor tissues, were collected at 24 h and 14 d post intravenous injection of RUCSNs-DM. After the organs were digested with nitric acid and hydrogen peroxide, Y element content of the various organs was measured by an inductively coupled plasma optical


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emission spectrometer (ICP-OES), as displayed in Figure 7D. The mice showed an effective accumulation of RUCSNs-DM in the tumor site 24 h after intravenous injection, which could be due to the enhanced penetration and retention (EPR) effect. Moreover, the accumulation of RUCSNs-DM in the reticuloendothelial system (RES), such as liver, spleen and lung, was found at the 24 h time point.51 Over time, the content of RUCSNs-DM in the major organs was gradually reduced (Figure 7D).

In vivo cancer therapy studies of RUCSNs-DM with efficient SO2 generation

Figure 8. In vivo tumor therapy by SO2 gas. (A) Relative tumor volumes changes, (B) survival curves, and (C) Body weights changes of mice in different groups during the therapy process. (D) HE and TUNEL stained histological images of tumor slices collected from different groups (control: treatment with PBS) of mice after treatment for 1 day. Scale bar: 50 μm. Data points represent mean ± s.d. (n = 5 mice per group).


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**P < 0.01, ***P < 0.005.

In vivo anti-tumor efficacy of SO2 gas was investigated in subcutaneous S180 tumor-bearing nude mice. The mice were randomly divided into six groups: 1) control (PBS), 2) NIR only, 3) DM, 4) RUCSNs, 5) RUCSNs-DM, and 6) RUCSNs-DM with NIR light irradiation. When the tumor size reached ~50 mm3, different formulations were intravenously injected into the tumor-bearing mice through the tail vein for four different times (day 0, 3, 6, and 9), followed by 980 nm laser irradiation at 24 h post each injection (1 W/cm2, 15 min). The tumor sizes and body weights of the mice were recorded every other day. The relative tumor volumes of mice in the control and NIRonly (980 nm, 1 W/cm2, 10 min) groups showed rapid growth with negligible difference from the control group, which revealed that the irradiation dose of NIR light employed in the experiments had little destructive effects on mice and tumors (Figure 8A). The mice in the RUCSNs group showed similar growth tendency and tumor sizes relative to these in the control group, indicating that single RUCSNs had no therapeutic effect. An insignificant inhibition of tumor growth was observed in the DM group, which can be attributed to the slight toxicity of DM after four-dose treatment. Furthermore, in the RUCSNs-DM without NIR irradiation group, the tumor sizes were close to those in the control group, implying that RUCSNs-DM had no obvious anti-tumor effect without NIR irradiation, and that the slight toxicity of DM could be reduced after loading in the RUCSNs (Figure S22). More significantly, the tumor growth of mice in the RUCSNsDM with NIR irradiation group was evidently suppressed and the growth inhibition rate


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of tumors was over 80% after four times treatment, demonstrating the high efficacy of NIR responsive SO2 therapy. Additionally, the 30-day-survival rate of mice in the RUCSNs-DM with NIR irradiation group reached 100%, which was much higher than that of mice in the other groups (Figure 8B), indicating that SO2 therapy could significantly prolong the survival time of tumor-bearing mice. In addition, no obvious fluctuation in mouse body weight was observed in all groups (Figure 8C), indicating no obvious side effects of these formulations. In order to gain insight into the therapeutic effects of the different treatments, tumor tissues of mice in the different groups were collected and processed for histological analyses. Based on the results of TUNEL staining assay of tumor sections (Figure 8D), a higher level of cell apoptosis was observed in the RUCSNs-DM treated with NIR irradiation group than that in the other groups, which proved the presence of DNA fragmentation, a characteristic feature of cell apoptosis. Therefore, the results suggested that the NIR responsive SO2 therapy of RUCSNs-DM was able to activate apoptosis in tumor tissues. Moreover, hematoxylin and eosin (H&E) staining assays also demonstrated that the tumor cells treated with RUCSNs-DM and NIR irradiation were seriously damaged, whereas the tumors of other groups had no distinct injury, proving the excellent anti-tumor effects of RUCSNs-DM with NIR laser irradiation. The other main organ sections (heart, liver, spleen, lung, and kidney) treated with different formulations were collected at the end of the treatment and based on the H&E staining assays, no detectable lesions were found compared to the control group (Figure S23).


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CONCLUSION In conclusion, a NIR light responsive SO2 gas therapy platform for highly efficient

in vivo anti-tumor therapy, based on RUCSNs-DM was developed. Benefiting from the prominent upconversion luminescence, UCNPs were selected as a photoconverter which can convert NIR photons to high-energy UV photons and efficiently activate DM for generating SO2 gas, overcoming the tissue penetration depth limitation of UV light for in vivo cancer therapy. Facilitated by the yolk-shell structure of the RUCSNs, DM can be loaded in the RUCSNs with a high loading capacity and low leakage potential. Additionally, RUCSNs-DM has good dispersion stability, low cytotoxicity, effective cell uptake, and lysosome escape ability, as well as high hematology safety and in vivo biocompatibility. Significantly, RUCSNs-DM is capable of generating enough SO2 gas under 980 nm laser stimulation which elevates intracellular ROS level, resulting in nucleus DNA damage and eventual cell apoptosis. Moreover, RUCSNs-DM demonstrated efficient tumor accumulation and robust NIR-triggered anti-tumor efficacy without engendering adverse effects. As a gas therapy platform, such NIRresponsive SO2 therapy provides a good example for highly localized gas anti-tumor therapy and may expedite the development of potent in vivo cancer therapies.

EXPERIMENTAL METHODS Chemicals. Yttrium acetate hydrate (Y(ac)3), Ytterbium acetate hydrate (Yb(ac)3), Thulium acetate hydrate (Tm(ac)3), oleic acid (OA), ammonium fluoride (NH4F),


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tetraethylorthosilicate (TEOS), Igepal CO-520, and polyvinylpyrrolidone (PVP, Mw=40000) were purchased from Sigma-Aldrich. 1-Octadecene (ODE) was purchased from Alfa. Sodium hydroxide (NaOH), ammonium hydroxide (NH3·H2O), ethanol, and methanol (MeOH) were purchased from Sinopharm chemical reagent co. LTD. Calcein-AM, propidium iodide (PI) and CCK-8 were purchased from Dojindo Molecular Technologies, Inc. All other chemicals were of analytical grade and were used as received from manufacturer. Ultrapure water acquired from a Millipore water purification system (18.2 MΩ resistivity) was used in all runs. Characterization. Transmission electron microscopy (TEM) images were performed on a FEI Tecnai G2 F30 microscope at 200 Kv and a Hitachi HT7700 Exalens microscope at 120 Kv. N2 adsorption-desorption isotherms and the pore size were obtained by a Micromeritics ASAP 2460 surface area and porosity analyzer (USA). X-ray diffraction (XRD) patterns were acquired by D/MAX-Ultima VI X-ray powder diffractometer (Rigaku Co., Japan). The hydrodynamic diameter of the samples were measured by a Zeta potential analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd., England). UV-vis absorption spectra were recorded using an Infinite M200 PRO Microplate Reader (Tecan Trading Co., Ltd, Switzerland) at room temperature. The emission spectra were measured by a steady state/transient fluorescence

spectrometer

FLS980

(Edinburgh

instruments,

England).

The

fluorescence images of cells were obtained by Confocal Laser Scanning Microscope (CLSM, Nikon, Japan). Preparation

of

NaYF4:Yb

(25%)/Tm

(0.3%)@NaYF4

(UCNPs).


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NaYF4:Yb(25%)/Tm (0.3%) core was firstly prepared as previously reported method with some modifications.38,

39

Briefly, 0.4 mmol of rare earth salts [Y(ac)3 (74.7%);

Tm(ac)3 (0.3%); Yb(ac)3 (25%)] were added into a 50 mL flask containing 3 mL OA and 7 mL ODE under vigorous stirring. The mixture was heated up to 150 °C for 30 min. After cooling to 50 °C, 2 mL 0.5 M NaOH in MeOH and 4 mL 0.4 M NH4F in MeOH were added and kept reacting for 35 min. Then, the mixed solution was degassed at 100 °C for 20 min to exclude redundant MeOH under the vacuum condition, and further heated up to 290 °C for 90 min. After cooling to room temperature with stirring, the product was obtained by centrifugation. In order to grow the inert NaYF4 outer shell, the mixed solution of 0.13 mmol Y(ac)3, 3 mL OA, and 7 mL ODE was vigorously stirred in a 50 mL flask and heated up to 150 °C for 30 min. After cooling to room temperature, the as-prepared NaYF4:Yb/Tm core was added and the mixed solution was heated up to 80 °C for 20 min. After cooling to 50 °C, NaOH and NH4F were added to the mixture solution. The following operations were carried out by reference to the synthesis of NaYF4:Yb/Tm core. Preparation of RUCSNs. RUCSNs were synthesized by a two-step method. In the first step, the UCNPs coated with amorphous silica shell were prepared according to the previous method.40, 41 In a typical method, 0.5 mL Igepal CO-520 was dispersed in 9 mL cyclohexane and reacted with cyclohexane solution of UCNPs (20 mg/mL) for 3 h at room temperature. Next, 0.75 mL 30% NH3·H2O was added to the above solution and the mixed solution was stirred for another 0.5 h. After addition of 150 μL TEOS, the solution was stirred for 24 h. The as-synthesized UCNP@SiO2 NP was


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collected by centrifugation, washed by ethanol for twice, and finally dispersed in deionized water. In the second step, in order to etch Igepal CO-520 template, PVP (0.2 g, Mw = 40000) was added into the aqueous solution of UCNP@SiO2 and stirred for 0.5 h at room temperature. Then, the mixture was heated to 96 °C for 6 h. The solution was then cooled down to room temperature and subjected to centrifugation to obtain RUCSNs. Preparation

of

RUCSNs-DM.

1-(2,5-dimethylthien-1,1-dioxide-3-yl)-2-(2,5-

dimethylthien-3-yl)-hexafluorocyclopentene (DM) was synthesized according to the reported method.52 For loading DM, RUCSNs (10 mg/mL) in ethanol was mixed with 5 mg DM and stirred for 24 h in dark. The RUCSNs-DM was obtained by centrifugation and washed by using ethanol. The loading efficiency of DM in RUCSNs was calculated

via a standard curve of DM measured by UV-Vis spectroscopy. NIR-responsive SO2 release experiment. In order to measure the NIR-responsive SO2 generation, the aqueous solutions of RUCSNs-DM were subjected to 980 nm laser irradiation (continuous wave, 1 W/cm2) for 0, 5, 10, 15, 20, 30, and 50 min, respectively. Subsequently, fresh 7-diethylaminocoumarin-3-aldehyde (DEACA) solution was added into the aqueous solutions of RUCSNs-DM. After further reaction for 10 min, the fluorescence intensity of each solution was measured by fluorescence spectrophotometer (Ex: 390 nm). Cell culture and cytotoxicity assay. HeLa, MCF-7, and S180 cells were cultured with DMEM medium (Hyclone) containing with 10% fetal bovine serum (FBS, Gibco)


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at 37 °C, 5% CO2. For cytotoxicity assay of RUCSNs, the seeded HeLa, MCF-7, and S180 cells were cultured overnight in 96-well plates, and then incubated with different concentrations of RUCSNs for 24 h at 37 °C. Cell viability was measured with CCK-8 kit referring to the manufacturer’s protocol. NIR-responsive in vitro cytotoxicity. HeLa cells were seeded in 96-well plates and cultured for 24 h (37 °C, 5% CO2). After incubating with different concentrations of RUCSNs-DM for 6 h, the medium was removed and replaced with fresh medium. Then, the HeLa cells were irradiated with 980 nm laser for 10 min (1 W/cm2), and incubated for another 16 h under dark. Cell viability was measured with CCK-8 kit. Cellular Calcein-AM and PI co-staining. In order to assess the therapy effect of SO2 gas in vitro, the seeded HeLa cells were incubated with 200 µg/mL RUCSNs-DM for 6 h. Following with 980 nm laser irradiation (1 W/cm2, 10 min) and further incubation for 18 h, HeLa cells were stained with Calcein-AM and PI kit before imaging by CLSM. Confocal imaging of intracellular ROS. For evaluating the intracellular ROS level, DCFH-DA was employed as a ROS-sensitive probe. DCFH-DA solution was freshly prepared according to previous report approach.34 After seeding for 24 h, the HeLa cells were incubated with RUCSNs-DM for 12 h, followed by 980 nm laser irradiation (1 W/cm2, 10 min). Then, the cells were washed with PBS for three times and stained with DCFH-DA before imaging by CLSM. Confocal imaging of TUNEL staining. The HeLa cells were firstly seeded on incubatedconfocal dish for 24 h followed by incubation with RUCSNs-DM (100 µg/mL)


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for 6 h. Then, the HeLa cells were exposed to 980 nm laser (1 W/cm2, 10 min) and incubated for another 16 h. In the next step, the cells were washed with PBS twice, and then fixed with 4% paraformaldehyde for 1 h. Finally, the cells were stained with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) agent (In Situ Cell Death Detection kit, Roche) and imaged by CLSM immediately. Flow cytometry of Annexin V-FITC and PI co-staining. After treatment with RUCSNs-DM solutions and NIR laser irradiation, the HeLa cells were collected and stained with Annexin V-FITC and PI immediately before analysis by flow cytometry (10000 cells for each experiment). Hemolysis Assay. The red blood cells (RBCs) were collected from the mice. After washing with PBS, the RBCs were treated with different concentrations of RUCSNsDM (0 to 1000 μg/mL) at room temperature for 2 h. The negative and positive controls were treated with PBS and deionized water, respectively. After centrifugation, the absorbance of the supernatants was recorded at 541 nm. The percent hemolysis was calculated (A: absorbance) by the equation: hemolysis (%) = ([Asample − Anegative] / [Apositive − Anegative) × 100 %.

In vivo biodistribution study. All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and approved by the Institutional Animal Care and Use Committee of Fujian Medical University. Tumor-bearing Balb/c mice model were prepared by hypodermic inoculation of S180 cells (1×106) in the right hind leg of mice.


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For studying the biodistribution of RUCSNs-DM in mice, the Y element contents in main organs (heart, liver, spleen, lung, kidney and tumor) were tested by ICP-OES. The tumor-bearing mice after intravenous of the samples were sacrificed to collect the major organs and tumor of the mice at post-injection of 24 h and 14 d. The issues were added into the mixed solution of hydrogen peroxide and nitric acid mixtures (1:6, v/v) and the mixtures were heated up to 120 °C for 24 h. After further centrifugation and filtration of the resulting solutions, the supernatants were diluted with 2% nitric acid for ICP-OES test to measure the contents of Y element.

In vivo therapy. For in vivo therapy, the S180 tumor-bearing mice were randomly divided into six groups with 5 mice per group. When the tumor volume grew up to 50 mm3, the mice were intravenously injected with different formulations every third day for four times. These groups were: (1) control (PBS), (2) NIR only, (3) DM, (4) RUCSNs, (5) RUCSNs-DM, and (6) RUCSNs-DM with NIR (dose: 20 mg/kg RUCSNs, 2 mg/kg DM). For each treatment, the mice of NIR only group and RUCSNs-DM with NIR group were irradiated with 980 nm laser (1 W/cm2) for 15 min at 24 h post-injection. The tumor size and weight were measured every two days.

ASSOCIATED CONTENT

Supporting Information Available: The Supporting Information file includes TEM and HRTEM images, the particle size distribution, and XRD patterns of NaYF4: Tm/Yb, TEM image of UCNPs@amorphous silica shell, diagram of the synthesis process of


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DM, 1H NMR of 2-iodine-1, 4-dimethylthiophene, DMTF6, and DM, UV-vis absorption spectrum of DM with different concentrations, standard work curve of DM, photograph and DLS distribution of RUCSNs-DM in different solutions, DLS distribution of RUCSNs-DM in different medium solutions during 12 days' storage, the release percentage of DM from the RUCSNs-DM at different pH, intracellular trafficking images of RUCSNs-DM studied by CLSM, The blood chemistry analysis of mice before and after injection for different times, representative photos of tumor-bearing mice before and after 16 days' treatment, and H&E stained images of major organs. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Jibin Song: [email protected] Xiaoyuan Chen: [email protected] Huanghao Yang: [email protected]

Author Contributions All authors have given approval to the final version of the manuscript.


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ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (Nos. U1505221, 21475026, 21874024), and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11).

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H.;

Shangguan,

D.;

Tan,

W.,

Facile

Phase

Transfer

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Near-Infrared Light-Triggered Sulfur Dioxide Gas Therapy of Cancer

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