Ultrasound is a mechanical wave with periodic vibrations in a continuous medium at frequencies greater than 20 kHz [12]. Ultrasound is able to penetrate tissue with less attenuation of energy. Therefore, it can be applied to medical diagnosis and therapeutic use. For a medical diagnosis purpose, the ultrasound is irradiated at a frequency of 2.0 to 28.0 MHz with low-energy irradiation to prevent tissue from damaging. For therapeutic use, the ultrasound is irradiated at a frequency of 0.5 to 3.0 MHz with higher doses of energy to generate the desired biological results [13]. For SDT, low-intensity ultrasound is used to induce the non-thermal and sono-chemical effects to activate sonosensitizer to cause the damage and even the death of tumor cells [14]. The non-thermal effect of ultrasound in SDT is cavitation that involves formation, growth and collapse of cavitation bubbles [7]. Under ultrasound irradiation, the static pressure of the aqueous solution decreases below the vapor pressure, and water may evaporate into gas bubbles. The cavitation bubbles nucleate in the presence of impurities or pre-existing bubbles in solution and oscillate in the phase under irradiation [15]. During the ultrasound irradiation, bubbles grow increasingly larger and stop growing when the static pressure equals the vapor pressure. They may start to break down from its weakest spot when the static pressure exceeds the vapor pressure, and then collapse (known as inertial cavitation) led to a highly concentrated energy release [16,17]. The released energy leads to the pyrolysis reaction of the water, which generate (•OH) and short light pulses (known as sonoluminescence) [18]. Sonoluminescence involves intense ultraviolet-visible light, which can excite sonosensitizer to generate ROS [19].

Sonosensitizers play a critical role in SDT that can enhance the effect of ultrasound. The development of sonosensitizers had grown swiftly in recent decades due to the known mechanisms of cell apoptosis for SDT [20]. The porphyrin-based sonosensitizers, such as photofrin, hematoporphyrin, 5-ALA (5-aminolevulinic acid) and chlorin-e6, are the most often used sonosensitizers in SDT research [7]. However, porphyrin-based sonosensitizers have phototoxicity on the skin that may affect both tumor cells and normal cells under a certain wavelength of light or energy irradiation in PDT studies, which means that this issue may also take place in SDT [20]. On the other hand, most sonosensitizers were hydrophobic and easy to aggregate in physiological condition, leading to a reduction in their ROS production [21]. Nonetheless, the development of nanoparticles shows a promising potential to solve these problems. Among many nanosensitizers, titanium dioxide (TiO2) has attracted the most attention due to its properties [21]. TiO2 is widely used in many territories based on low toxicity, high stability, high photocatalytic activity and low cost [22,23]. Compared to porphyrin-based sonosensitizers which are quickly degraded under oxidizing conditions, TiO2 exhibits high stability because it is highly resistant to degradation by ROS. TiO2 exhibits three kinds of crystal structures, namely anatase, rutile and brookite. Anatase and rutile are the most common in the utilization of crystal structures, and brookite is less used in industrial application. Even though anatase (Eg = 3.2 eV) has a wider bandgap than rutile (Eg = 3.0 eV), anatase shows higher photoactivity due to its larger specific surface area that anatase is more suitable to be used as a photocatalyst [24]. In previous studies, the anatase structure of TiO2 has been utilized as a sonocatalyst to generate ROS under ultrasound irradiation [10,14]. Nonetheless, the wide bandgap of anatase requires a greater energy to trigger. Carbon has previously been doped in the semiconductors to form a new valence band, thus narrowing the bandgap [25]. The addition of carbon may give TiO2 an excess of conducting electrons or holes which is important for lowering the bandgap [23,24].

Hence, in this study, the sonosensitizer C-doped TiO2 was synthesized through doping carbon into the anatase structure of TiO2 to diminish the bandgap. A square wave of the ultrasound at a resonant frequency of 1.0 MHz, intensity of 0.33 MPa and duty cycle of 50% was used to induce the inertial cavitation and generate sonoluminescent light to activate the synthesized C-doped TiO2.

#### **2. Materials and Methods**

#### *2.1. Preparation of C-Doped TiO2*

C-doped TiO2 was synthesized by the sol-gel method [23]. First, 2 g of Pluronic® F127 (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 40 mL 95% ethanol completely with vigorous stirring. Then, 5 mL titanium(IV) isopropoxide (TIP, Ti(OCH(CH3)2)4, purity > 97%, Sigma-Aldrich) was added into the solution with magnetic stirring of 600 rpm. An amount of 3 g of D-(+)-glucose (C6H12O6, Sigma-Aldrich), which was used as the carbon source, was dissolved in 6 mL ddH2O. Then, the glucose solution was dropped into the TIP/Pluronic® F127 solution. The mixed solution was kept vigorously stirring at room temperature for 30 min. The precipitate was collected by centrifugation at 5000 rpm and washed with 95% ethanol for three times. The precipitate was then calcined at 400 ◦C for 2 h to obtain particles.

#### *2.2. Material Characterization*

The crystal structure of the synthesized C-doped TiO2 was determined by X-ray diffraction (XRD; Rigaku TTRAX 3) with Cu Kα radiation at a speed of 2◦ per minute at 40 kV and 30 mA from 20◦ to 60◦.The surface morphology and particle size of C-doped TiO2 were characterized by a scanning electron microscope (SEM; Nova Nano SEM 450). The structure and diffraction pattern of the material were analyzed by a transmission electron microscope (TEM; JEOL 2010F). The particle size of the material was reconfirmed via Zetasizer (Malvern Nano ZS). The chemical composition of the material was analyzed by an energy dispersive spectrometer (EDS; Nova Nano SEM 450) SEM attachment. To confirm that carbon had been doped in titanium dioxide and formed the bonding, C-doped TiO2 was measured by auger electron spectroscopy (AES; JEOL JAMP 9510F) and X-ray photoelectron spectroscopy (XPS; Theta Probe).

#### *2.3. Ultrasound Apparatus*

Ultrasound irradiation was conducted with a function generator (Agilent 33521A) at a resonant frequency of 1.0 MHz and a duty cycle of 50% and amplified by a power amplifier (E&I 1040L) to generate a square wave with a negative pressure of 0.33 MPa and intensity of 1.8 W/cm2 for 90 s in the whole experiment. Figure 1 shows the entire apparatus.

**Figure 1.** Ultrasound apparatus. The ultrasound transducer with a diameter of 2.0 cm was fixed with aluminum block by a rubber band to keep the transducer facing upward in the degassed water tank.

#### *2.4. Preparation of Terephthalic Acid Solution and Evaluation of Ultrasound Parameter*

First, 2 mM terephthalic acid (TA; Sigma-Aldrich) was dissolved in 800 mL ddH2O. During ultrasound treatment, the experiments should be carried out under alkaline condition. Hence, 5 mL 1 M of NaOH was added in the solution. TA solution was stirred for 1 h under 4 ◦C and dark conditions to avoid photochemical reaction [26].

The experiment was divided into two groups. No US group was named as the control group: the US group was the group irradiated by ultrasound. The fluorescence signal intensity was measured by a multi-label plate reader with excitation and emission wavelengths at 310 and 420 nm, respectively.

#### *2.5. Cell Culture and Animal Model*

Briefly, L929 cells obtained from National Health Research Institutes were cultured in MEM medium (Minimum Essential Media, Sigma-Aldrich) with 10% Fetal bovine serum (FBS, Gibco) and 1% Antibiotic-Antimycotic (Gibco) and maintained in a 5% CO2 atmosphere at 37 ◦C.

Breast cancer 4T1 cells obtained from National Health Research Institutes were used as model cancer cells in this study. The 4T1 cells were cultured in RPMI medium (RPMI-1640 Media (Sigma-Aldrich) with 10% Fetal bovine serum (FBS, Gibco) and 1% Antibiotic-Antimycotic (Gibco) and maintained in 5% CO2 atmosphere at 37 ◦C.

6-week-old male BALB/c nude mice were used as a xenograft animal model in this study. The nude mice were injected subcutaneously with 2 × 106 4T1 cells suspended in 200 μL 1X PBS into the right thigh. All the animal experiments were approved by National Health Research Institutes (NHRI-IACUC-107013).

#### *2.6. Evaluation of Biocompatibility for the Synthesized C-Doped TiO2*

The evaluation of biocompatibility in this study was based on International Standard ISO 10993. The cell viability induced by the synthesized C-doped TiO2 was determined by the WST-1 test (TAKARA). L929 cells were seeded on a 96-well cell culture plate with a cell density of 104 cells per well and incubated in 5% CO2 atmosphere at 37 ◦C. Briefly, 0.2 g/mL of the materials was immersed in MEM medium for 16 to 24 h in 5% CO2 atmosphere at 37 ◦C as material extracts. The control group included L929 cells without treatment. Zinc diethyldithiocarbamate (ZDEC, Sigma-Aldrich) and aluminum oxide (Sigma-Aldrich) were indicated as the "Positive Control" and "Negative Control" group, respectively. The C-doped TiO2 group included cells cultured with C-doped TiO2. After one-day incubation, the previous solution in each well was removed, and 100 μL of the supernatant of the material extracts was added into each well. After one-day incubation, the previous solution in each well was removed, and 100 μL fresh MEM medium with 10% WST-1 reagent was added. After 2 h incubation in 5% CO2 atmosphere at 37 ◦C kept in the dark, the solution in each well was collected to measure absorbance at 490 nm by a microplate reader.

#### *2.7. Detection of Cellular ROS Generation*

ROS generation induced by the synthesized C-doped TiO2 in SDT was measured by a DCFDA-cellular ROS detection assay kit (abcam). 4T1 breast cancer cells were seeded on 12-well cell culture plates with a cell density of 6 × 104 cells per well and incubated in 5% CO2 atmosphere at 37 ◦C. After one-day incubation, the previous solution in each well was removed, and 1 mL 15 mg/mL synthesized C-doped TiO2 in RPMI medium was added into each well. After 6 h incubation, ultrasound was then irradiated from the bottom of the cell culture plates in degassed water. The distance between ultrasound transducer and the bottom of the cell culture plate was around 5 mm. After US treatment, the cell culture plates were incubated for 2 h. The previous solution in each well was then removed and washed once with 1× PBS. An amount of 1 mL 20 μM 2 ,7 –dichlorofluorescin diacetate (DCFDA, abcam) in 1× buffer was added into each well for 30 min in 5% CO2 atmosphere at 37 ◦C in the dark. The previous solution in each well was removed, and cells were washed with 1× PBS once. An amount of 1 mL 1× PBS was added into each well. The fluorescence signal intensity was measured by a multilabel plate reader with excitation and emission wavelengths at 485 and 535 nm, respectively [27]. The control group included 4T1 cells without treatment. The C-doped TiO2 group included cells cultured with C-doped TiO2. The US group included cells subjected to ultrasound irradiation. The SDT group included cells cultured with C-doped TiO2 and subjected to ultrasound irradiation.

#### *2.8. Evaluation of the Synthesized C-Doped TiO2 in SDT In Vitro*

WST-1 and LDH tests were used to evaluate the cell viability and cytotoxicity of the synthesized C-doped TiO2 in SDT. In vitro, 6 × 104 4T1 cells suspended in 1 mL RPMI medium were seeded on 12-well cell culture plates and incubated in 5% CO2 atmosphere at 37 ◦C. After one-day incubation, the previous solution in each well was removed, and 1 mL 15 mg/mL synthesized C-doped TiO2 in RPMI medium was added into each well. After 6 h incubation, ultrasound was then irradiated from the bottom of the cell culture plates in degassed water. The distance between the ultrasound transducer and the bottom of the cell culture plate was around 5 mm. After ultrasound treatment, the cell culture plates were incubated for one day. For the WST-1 test, the previous solution in each well was removed, and 400 μL fresh RPMI medium with 10% WST-1 reagent was added. After 1 h incubation in 5% CO2 atmosphere at 37 ◦C kept in the dark, the solution in each well was collected to measure absorbance at 490 nm by an Elisa reader. The control group included 4T1 cells without treatment. Zinc diethyldithiocarbamate (ZDEC) and aluminum oxide were indicated as the "Positive Control" and "Negative Control" group, respectively. The C-doped TiO2 group included cells cultured with C-doped TiO2. The US group included cells subjected to ultrasound irradiation. The SDT group included cells cultured with C-doped TiO2 and subjected to ultrasound irradiation.

For the LDH test, 50 μL lysis solution was then added into each well of the Lysis group and then incubated in 5% CO2 atmosphere at 37 ◦C for 30 min. Fifty μL of supernatant from each well was then transferred to a 96-well cell culture plate, and 50 μL LDH reagent was added into each well. After 15 min of reaction at room temperature in the dark, the cytotoxicity was measured by a microplate reader with absorbance at 490 nm. The C-doped TiO2 group included cells cultured with C-doped TiO2. The US group included cells subjected to ultrasound irradiation. The SDT group included cells cultured with C-doped TiO2 and subjected to ultrasound irradiation.

#### *2.9. Evaluation of SDT in In Vivo Tumor Growth*

There were 4 different treatments exerted on nude mice: (1) the control group was injected with 200 μL 1× PBS at day 0 and day 7 without ultrasound irradiation; (2) the C-doped TiO2 group was injected with 200 μL synthesized C-doped TiO2 (150 mg/mL) in 1× PBS at day 0 and day 7 without ultrasound irradiation; (3) the US group was injected with 200 μL of 1× PBS at day 0 and day 7 with US irradiation; (4) the SDT group was injected with 200 μL synthesized C-doped TiO2 (150 mg/mL) in 1× PBS at day 0 and day 7 with US irradiation. There were 5 nude mice in each group.

The tumor volume was measured by an electronic slide caliper using Equation (1), where L is the longest dimension and W is the shortest dimension, parallel to the mouse body [28].

$$\mathbf{V} = \mathbf{0}.5 \times \mathbf{L} \times \mathbf{W}^2 \tag{1}$$

#### *2.10. Histopathology*

During sacrifice, cardiac puncture was used to obtain the blood samples. The samples were collected into tubes with 7.5% EDTA solution. Tumor and internal organs, including heart, liver, spleen, lung and kidney were also harvested and then fixed with 4% formaldehyde. These organs were embedded into the paraffin and further stained with hematoxylin and eosin (H&E) for histologic examination.

#### *2.11. Statistics*

All data are indicated as the mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons with the Dunnett test, and the difference between the groups was deemed to be statistically significant when *p* < 0.05.

#### **3. Results**

The XRD pattern of the synthesized C-doped TiO2 is shown in Figure 2a. It is indicated that the 2θ at 25.2◦, 38.3◦, 48◦, 54.3◦ and 55.1◦ corresponded to the crystal form of (101), (004), (200), (105) and (211), which was completely matched with the standard pattern of the anatase structure of TiO2 (JCPDS Card No. 21-1272). The surface morphology of C-doped TiO2 was characterized by SEM as shown in Figure 2b,c. Figure 2d shows the chemical composition of the material analyzed by EDS, Ti, O and C elements with an atomic percent (At%) of 62.8, 32.8 and 4.4, respectively. The particles were spherical with a rough surface. The structure and diffraction pattern of the material were examined by TEM as shown in Figure 2e,f, respectively. C-doped TiO2 formed in the aggregation of nanograins, and its particle size was in the range of 100 to 200 nm. The selected area diffraction pattern (SADP) of C-doped TiO2 showed a ring pattern with the concentric rings from interior to outside, representing the crystal planes of (101), (004) and (105) which corresponded to the anatase phase. The particle size was precisely measured by Zetasizer as shown in Figure 2g. The precise particle size was 156.9 nm with a polydispersity index (PDI) of 0.137, which was in agreement with the analysis of SEM particle size (148.9 ± 26.3 nm) as shown in Figure 2h. Figure 3a shows the detection of chemical composition of the material by AES. In addition to the peaks related to Ti and O, C was detected at the energy of 263 eV. Figure 3b shows the XPS spectra of C-doped TiO2 was at a binding energy of 282, 462 and 562 eV, representing O 1s, Ti 2p and C 1s, respectively. Figure 3c shows the XPS spectra of the C 1s scan: the peak of TiC was detected at binding energy around 282 eV [29]. Figure 3d shows the XPS spectra of the Ti 2p scan, the peak of Ti4+ was detected at a binding energy of 462 (Ti 2p1/2) and 456 eV (Ti 2p3/2). Figure 3e shows the XPS spectra of the O 1s scan: the peak of (Ti4+/Ti3+)-O was detected at a binding energy of 527 eV.

**Figure 2.** *Cont*.

**Figure 2.** (**a**) XRD pattern of C-doped TiO2, (**b**) low-magnification SEM images of C-doped TiO2, (**c**) high-magnification SEM images of C-doped TiO2, (**d**) EDS of C-doped TiO2, (**e**) TEM images of C-doped TiO2, (**f**) Diffraction pattern of C-doped TiO2, (**g**) Size distribution of C-doped TiO2 and (**h**) SEM particle size distribution of C-doped TiO2.

**Figure 3.** (**a**) AES spectra of C-doped TiO2, (**b**) XPS spectra of C-doped TiO2, (**c**) C 1s scan, (**d**) Ti 2p scan and (**e**) O 1s scan.

The 2-hydroxyterephthalic acid (HTA) fluorescence emission of the TA solution with or without US irradiation is shown in Figure 4. TA is a non-fluorescent compound and may further react with hydroxyl radicals to produce a highly fluorescent HTA. The fluorescence was 106 ± 4% (US group) and 100 ± 2% (control group), respectively. The fluorescence emission of the control group was 100% as the baseline. Each experiment was repeated 6 times. Based on the result, we believe that the US parameter in this study is able to induce inertial cavitation.

**Figure 4.** Oxidation of terephthalic acid (TA) to 2-hydroxyterephthalic acid (HTA) in the presence of hydroxyl radicals induced by inertial cavitation (one-way ANOVA, mean ± SD, *n* = 6, \*\*: *p* < 0.01).

The biocompatibility test of C-doped TiO2 on cell viability by the WST-1 test is shown in Figure 5. After treatment with C-doped TiO2, the cell viability was 0.25 ± 0.24% (positive control group), 99.54 ± 10% (negative control group), 100 ± 24% (control group) and 81.49 ± 20% (C-doped TiO2 group), respectively. The cell viability of the control group was 100% as the baseline. Each experiment was repeated 5 times. The biocompatibility test followed by the regulations of ISO 10,993 indicated that there would be no potential toxicity when the cell viability of the experiment group was over 75%. Based on the WST-1 test, C-doped TiO2 showed no potential toxicity to normal cells.

**Figure 5.** WST-1 test for the biocompatibility of C-doped TiO2 (one-way ANOVA, mean ± SD, *n* = 5, \*\*\*\*: *p* < 0.0001).

ROS generation of C-doped TiO2 under US irradiation by the DCFDA test is shown in Figure 6. DCFDA was able to permeate into 4T1 cells and be deacetylated by cellular esterases to a non-fluorescent DCFH. DCFH was then oxidized to a highly fluorescent 2 , 7 –dichlorofluorescein (DCF) in the presence of ROS. After the treatments, the fluorescence was 100 ± 7% (control group), 97 ± 12% (C-doped TiO2 group), 119 ± 13% (US group) and 141 ± 11% (SDT group), respectively. The fluorescence of the control group was 100% as the baseline. Each experiment was repeated 6 times.

**Figure 6.** DCFDA test for relative ROS concentration induced by C-doped TiO2 activated by US (one-way ANOVA, mean ± SD, *n* = 6, \*: *p* < 0.05, \*\*\*\*: *p* < 0.0001).

The effect of C-doped TiO2 in SDT on cell viability and cytotoxicity of 4T1 cells was examined by WST-1 and LDH tests. The results of live/dead assay are shown in Figure S1 to evaluate cell viability in C-doped TiO2 during ultrasound irradiation. After the treatments, the cell viability was 0 ± 1% (positive control group), 90 ± 13% (negative control group), 100 ± 14% (control group), 84 ± 3% (C-doped TiO2 group), 77 ± 2% (US group) and 67 ± 7% (SDT group), respectively, as shown in Figure 7a. The cell viability of the control group was 100% as the baseline. Each experiment was repeated 6 times. After the treatments, the cytotoxicity was 100 ± 18% (total lysis group), 0 ± 3% (C-doped TiO2 group), 7 ± 13% (US group) and 23 ± 13% (SDT group), respectively, as shown in Figure 7b. The cytotoxicity of the control group was 0% as the baseline.

The antitumor efficacy of C-doped TiO2 under ultrasound irradiation was evaluated using BALB/c nude mice subcutaneously injected with 4T1 cells, where C-doped TiO2 particles were delivered into mice during the entire experiment and then treated with ultrasound irradiation twice—on day 0 and day 7 (Figures S2 and S3). The variation of relative tumor volume is shown in Figure 8, which was used to evaluate the antitumor effect of C-doped TiO2 in SDT for 4T1 cells. At 14 days, the relative tumor volume was 367 ± 176% (control group), 407 ± 73% (C-doped TiO2 group), 335 ± 82% (US group) and 165 ± 22% (SDT group), respectively. The H&E-stained images of organs are shown in Figure S4. The result showed that the C-doped TiO2 group and US group could not suppress the growth of the tumor. On the other hand, the SDT group could retard the tumor growth significantly (*p* < 0.05). The histologic staining of the tumor site is shown in Figure 9. H&E-stained tumor tissue images confirmed that SDT enhanced the ability to cause the death of the 4T1 cells compared to the other groups.

**Figure 7.** (**a**) Cell viability induced by C-doped TiO2 activated by US evaluated by WST-1 assay (one-way ANOVA, mean ± SD, *n* = 6), (**b**) cytotoxicity induced by C-doped TiO2 activated by US evaluated by LDH assay (one-way ANOVA, mean ± SD, *n* = 6, \*: *p* < 0.05, \*\*\*: *p* < 0.001, \*\*\*\*: *p* < 0.0001).

**Figure 8.** The variation of relative tumor volume in BALB/c nude mice induced by C-doped TiO2 activated by US (one-way ANOVA, mean ± SD, *n* = 5, \*: *p* < 0.05, \*\*: *p* < 0.01).

**Figure 9.** H&E-stained images of tumor tissue. (**a**) Control group, (**b**) C-doped TiO2 group, (**c**) US group and (**d**) sonodynamic therapy (SDT) group.

#### **4. Discussion**

TiO2 is one of most representative material studied in inorganic sonosensitizers. Nonetheless, the low quantum yield of ROS limits the effectiveness of TiO2 as a sonosensitizer by rapid recombination of the free electron and electron hole. The addition of noble metals, such as Pt and Au, have been reported to retard carrier combination [30,31]. Pt-doped or Au-doped TiO2 has been confirmed to show therapeutic efficacy and suppress the growth of tumors significantly. However, the price of novel metals would increase the cost of material preparation dramatically. Carbon shows highly promising dopant to narrow the bandgap, reaching similar therapeutic efficacy to a novel metal-based system in a more economical way. The comparative table is listed in Table 1.

**Table 1.** The sonosensitizers and the matched ultrasound parameters.

In Figure 2a, the crystal structure of synthesized C-doped TiO2 was matched with the standard pattern of the anatase structure without significant structure change when carbon atoms played the substitutional foreign atoms, replacing some of the oxygen atoms. The results corresponded with the previous study [35]. In Figure 3c, the peak at around 282 eV was ascribed to Ti-C bonds. Thus, the Kröger–Vink notation of possible substitution of O2− by C was proposed and shown in Equation (2). The creation of oxygen vacancies may form the impurity band that contributes a band tailing effect, thus diminishing the bandgap [36,37]. In the previous study [38], comparing the typical Ti-O binding energy, the clear blue-shift was shown, which indicated the presence of oxygen vacancy after the reduction.

$$\text{TiC} \stackrel{\text{TiO}\_2}{\rightarrow} \text{Ti}^{\times}\_{\text{Ti}} + \text{Co}^{\prime\prime} + \text{Vo}^{\bullet\bullet} \tag{2}$$

In Figure 2c,g, the particle size of C-doped TiO2 was 156.9 nm which made the nanoparticles possible to accumulate in tumor tissue without entering the normal tissue [39].

Mechanical index (MI) was one of the indicators for evaluating the occurrence of the inertial cavitation. MI was calculated using Equation (3), where P- is the peak-negative acoustic pressure and *fc* is the center frequency of the ultrasound transducer. It was shown that 0.3 MPa of the peak-negative acoustic pressure was the least pressure to induce the inertial cavitation in aqueous solution [40]. In Figure 4, under US irradiation, the production of HTA increased significantly compared to the control group. In other words, US irradiation is able to elevate the OH generation in TA solution. Hence, the US parameter in this study was confirmed to be effective to induce the inertial cavitation.

$$\text{MII} = \frac{P}{\sqrt{f\_c}}\tag{3}$$

In Figure 6, compared to the control group, 4T1 cells treated with C-doped TiO2 did not increase the level of ROS; 4T1 cells treated with US could be effective to increase the level of ROS, ascribed to the pyrolysis reaction of the water to produce hydroxyl radicals; 4T1 cells treated with C-doped TiO2 plus US could increase the most significant level of ROS than the other groups, ascribed to the highly reactive singlet oxygen and hydroxyl radicals. This result corroborated that, under US irradiation, ROS generation could be increased in the presence of C-doped TiO2. However, the amount of extracellular generation of ROS may affect the cell toxicity of tumor cells. Figure 7 shows that SDT induced higher

toxicity in 4T1 cells than the other three groups. The results show that only US treatment seemed to cause insufficient ability to damage the 4T1 cells with the production of hydroxyl radicals. Nonetheless, US treatment in the presence of C-doped TiO2 seemed to significantly suppress the proliferation of 4T1 cells. The cellular uptake of C-doped TiO2 to 4T1 cells was supposed to occur via endocytosis or pinocytosis [41]. The possible pathway of cell damage induced by SDT may be speculated as: Under US irradiation, C-doped TiO2 was activated by the sonoluminescent light, induced by inertial cavitation, to an excited state. Carbon was doped in the anatase TiO2 to form a new valence band, thus narrowing the bandgap. The addition of carbon may give TiO2 an excess of conducting electrons or holes, which is important for lowering the bandgap [23,24]. The unstableness of the excited state may cause decay back to the ground state, leading to energy release. The released energy may transfer to oxygen to generate the highly reactive singlet oxygen and water to generate hydroxyl radicals (Figure 10). With the increasing concentration of singlet oxygen and hydroxyl radicals, 4T1 cells would be gradually damaged and lead to cell senescence and death due to oxidative damaging effects [11,42,43]. Due to the enhanced permeability and retention (EPR) effect, C-doped TiO2 preferentially accumulates in tumor cells eliciting efficient ROS generation [44] and further increasing the effectiveness of the SDT treatment. It was also revealed that the suppression of the tumor is due to the elevated level of ROS which results in both direct tumor cell death and blood vessel stasis. ROS can induce blood stasis via platelet aggregation or vessel constriction by destroying the endothelial layer [45]. Furthermore, SDT may elevate the level of inflammatory-associated cytokine (including TNF-alpha, IL-6, IL-1) production which is known to stimulate the maturation and function of granulocytes and macrophages [46].

**Figure 10.** The possible pathway of 4T1 tumor cell damage induced by SDT.

To further investigate the cell death of 4T1 cells induce by SDT, in-vivo study was implemented in this study. Figure 8 shows that SDT could be more effective in suppressing the growth of the 4T1 tumor with a significant difference between SDT and the other groups on day 7. The relative tumor volume of SDT ended with almost half compared to the other groups at the 14th day of the treatment. H&E-stained tumor tissue is shown in Figure 9. In the tumors treated with 1× PBS or C-doped TiO2 individually, integral tumor cellularity with spindle shape nuclei was visible. In the tumors treated with US, attributed to the inertial cavitation effect of US, a small area of nuclear fragmentation with little necrosis was visible. However, in the tumors treated with SDT, with the synergistic effect of hydroxyl

radicals and singlet oxygen, a huge necrotic area with shrinkage and fragments of nuclei was seen. These results corroborated that SDT could inhibit the proliferation of 4T1 tumor cells.

#### **5. Conclusions**

The availability of C-doped TiO2 nanoparticles as sonosensitizers activated by low-intensity ultrasound to generate ROS for cancer treatment was investigated in this study. Spherical C-doped TiO2 nanoparticles with an average particle size of around 157 nm were synthesized by the sol-gel process. From the WST-1 test, C-doped TiO2 showed no potential toxicity to normal cells. From in vitro study, it showed more extracellular generation of ROS in 4T1 breast cancer cells under SDT (C-doped TiO2/US treatment). Moreover, the effect of C-doped TiO2 in SDT could reduce the cell viability and increase the cytotoxicity of 4T1 cells. From in vivo study, the antitumor effect of C-doped TiO2 in SDT may inhibit the proliferation of the 4T1 tumor and lead to a huge necrotic area with shrinkage and fragments of nuclei in the tumor site. We believe that C-doped TiO2 nanoparticles could be viewed as sonosensitizers in SDT activated by low-intensity ultrasound for ROS generation as an effective strategy for alternative cancer treatments in the future.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/9/880/s1, Figure S1: Live/Dead assay; Figure S2: survival rate of BALB/c nude mice; Figure S3: average weight of BALB/c nude mice; Figure S4: H&E-stained images of organs.

**Author Contributions:** Conceptualization, C.-C.Y. and F.-H.L.; methodology, G.-S.C. and C.-H.H.; software, C.-X.W.; validation, C.-Y.K., C.-Y.C. (Chih-Ying Chi), C.-Y.C. (Ching-Yun Chen) and Y.-Y.L.; formal analysis, C.-X.W.; investigation, C.-Y.K., C.-Y.C. (Chih-Ying Chi), C.-Y.C. (Ching-Yun Chen) and Y.-Y.L.; resources, C.-H.H. and F.-H.L.; data curation, C.-C.Y.; writing—original draft preparation, C.-X.W.; writing—review and editing, C.-C.Y.; visualization, C.-X.W.; supervision, C.-H.H., G.-S.C., and F.-H.L.; project administration, F.-H.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Water Soluble Iron-Based Coordination Trimers as Synergistic Adjuvants for Pancreatic Cancer**

**Marco Cordani 1, Esther Resines-Urien 1, Arturo Gamonal 1, Paula Milán-Rois 1, Lionel Salmon 2, Azzedine Bousseksou 2, Jose Sanchez Costa 1,\* and Álvaro Somoza 1,\***

**Abstract:** Pancreatic cancer is a usually fatal disease that needs innovative therapeutic approaches since the current treatments are poorly effective. In this study, based on cell lines, triazole-based coordination trimers made with soluble Fe(II) in an aqueous media were explored for the first time as adjuvant agents for the treatment of this condition. These coordination complexes were effective at relatively high concentrations and led to an increase in reactive oxygen species (ROS) in two pancreatic cancer cell lines, PANC-1 and BXPC-3, and this effect was accompanied by a significant reduction in cell viability in the presence of gemcitabine (GEM). Importantly, the tested compounds enhanced the effect of GEM, an approved drug for pancreatic cancer, through apoptosis induction and downregulation of the mTOR pathway. Although further evaluation in animal-based models of pancreatic cancer is needed, these results open novel avenues for exploring these iron-based materials in biomedicine in general and in pancreatic cancer treatment.

**Keywords:** pancreatic cancer; antitumor agents; coordination polymers; bioinorganic chemistry; reactive oxygen species

#### **1. Introduction**

Pancreatic ductal adenocarcinoma (PDAC) is an orphan disease with a terrible prognosis, even when diagnosed early. The survival rate after five years remains below 5%, despite tremendous efforts at the preclinical and clinical stages. Worldwide, in 2018 there were 460,000 new cases of pancreatic cancer and 430,000 associated deaths [1]. The early symptoms of the disease are extremely rare, and, therefore, most patients present with locally advanced disease and/or metastasis at the time of diagnosis, limiting their options of being treated by surgery [2]. The standard treatment for the advanced disease includes gemcitabine (2 ,2 -difluoro-2 -deoxycytidine; GEM), with a response rate of less than 20% [3]. Therefore, the identification of novel therapeutic strategies to overcome the limitations of current therapies against PDAC is urgent [4].

In this regard, we are exploring the use of coordination polymers (CPs) as adjuvants for the treatment of pancreatic cancer. These materials are built from metal ions linked by ligands, forming different topologies ranging from 1D to 3D structures. A great variety of CPs have been prepared with different properties and functionalities, such as porous frameworks for gas sorption and separation, catalysis, molecular and ion-exchange, molecular magnets, luminescence, among others [5]. The biological applications of these materials have been limited mainly due to their low solubility in biological media. Nevertheless, they are extremely appealing due to the versatility that confers the use of different biocompatible metals or ligands [6]. Thus, CPs have been employed as nanocarriers [7–11], to control the

**Citation:** Cordani, M.; Resines-Urien, E.; Gamonal, A.; Milán-Rois, P.; Salmon, L.; Bousseksou, A.; Costa, J.S.; Somoza, Á. Water Soluble Iron-Based Coordination Trimers as Synergistic Adjuvants for Pancreatic Cancer. *Antioxidants* **2021**, *10*, 66. https:// doi.org/10.3390/antiox10010066

Received: 16 December 2020 Accepted: 3 January 2021 Published: 7 January 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

release of therapeutics [12–15], as contrast agents in MRIs [16,17] or optical imaging [18], and even in theranostic approaches [6].

A particularly interesting family of iron CPs is the chain-like polymeric Fe(II) system, with general formula {[Fe(Rtrz)3]X2}n, (Rtrz = substituted triazole; X = monovalent anion), which have been the focus of remarkable studies in a solid state [19]. These Fe(II) systems present the physical phenomenon of spin-crossover, which is accompanied by a strong thermochromism and a magnetic transition between diamagnetic and paramagnetic behavior in the solid state either as powder [20,21] or as nanoparticles [22]. However, less attention has been paid to the phenomenon in an aqueous solution [23], mainly due to their poor solubility [24]. To overcome this limitation, shorter polymeric chains (more precisely, trinuclear oligomers) are used in this report, providing water-soluble derivatives.

Reactive oxygen species (ROS) are highly reactive molecules containing oxygen produced by cellular metabolism, and they participate in several biological processes, including signal transduction, enzyme activation, gene expression, and protein post-translational modifications [25]. However, although the role of ROS in cellular physiology is well established, when produced in excess, they may cause irreversible cellular damage through the oxidation of biomolecules such as lipid membranes, enzymes, or DNA, which generally leads to cellular death [26]. ROS can also lead to carcinogenesis and tumor progression by inducing DNA mutations, genomic instability, and pro-oncogenic signaling pathways [27]. In this regard, cancer cells frequently exhibit increased ROS, mainly resulting from their high metabolic activity and oncogene activation [27]. However, this increased oxidative stress can promote cell proliferation without leading to cell death, but may make cancer cells vulnerable to exogenous oxidizing agents that generate additional ROS, which may increase oxidative stress levels above the cytotoxic threshold [28]. For this reason, the overstimulation of ROS by anticancer agents has been largely exploited and represents a main mechanism through which chemotherapy drugs kill cancer cells [29,30].

In this regard, once the oligomers were synthesized and characterized, their effect on cell viability and ROS induction, in the presence or absence of GEM, were assessed in two pancreatic cancer cell lines (PANC-1 and BXPC-3) as well as in a non-tumoral cell line (HaCaT). Furthermore, the oligomer's (or CP's) effect on apoptosis and the mTOR pathway within pancreatic cancer cells was evaluated to dissect the molecular and functional aspects underlying the mechanism of action of such structures. Finally, the involvement of ROS in the regulation of cell viability and the mTOR pathway was analyzed. Overall, this study provides new insight into the molecular mechanisms of action of CPs, which could be exploited as adjuvants for the treatment of pancreatic cancer.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Roswell Park Memorial Institute (RPMI) medium, Dulbecco's Modified Eagle's (DMEM) medium, streptomycin–penicillin (100×), fetal bovine serum (FBS), l-glutamine (100×), trypsin (10×), phosphate-buffered saline (PBS), and cell culture plasticware were purchased from VWR. Gemcitabine was purchased from Fluorochem. *N*-acetyl cysteine was purchased from Sigma Aldrich, Saint Louis, MO, USA. Chemicals and reagents were purchased from commercial suppliers and used as received following the indications reported.

#### *2.2. Cell Lines and Culture Conditions*

PANC-1 and HaCaT cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM medium with 10% FBS, 1% streptomycin–penicillin, and 1% l-glutamine at 37 ◦C in a Binder CB210 incubator (5% CO2). BXPC-3 was a gift from Ibane Abasolo Olaortua (CIBBIM-Nanomedicine Institut de Recerca Hospital Universitari Vall d'Hebron) and was cultured in RPMI medium with 10% FBS, 1% streptomycin–penicillin, and 1% l-glutamine at 37 ◦C in a Binder CB210 incubator (5% CO2). All the procedures were performed inside a laminar flow hood Telstar CV-30/70 (Telstar, Terrassa, Spain).

#### *2.3. Analysis of ROS*

The non-fluorescent diacetylated 2 ,7 -dichlorofluorescein (DCF-DA) probe (Sigma Aldrich, Saint Louis, MO, USA), which becomes highly fluorescent upon oxidation, was used to evaluate ROS production.

The ability of trimers to generate ROS in free-cell water was assessed after their incubation at three different concentrations (1 μM, 100 nM, or 1 nM) with 1 μM DCF-DA for 48 h. Salt and hydrogen peroxide were incubated with the probe as control. DCF-DA fluorescence was measured by using a multimode plate reader (λexc = 485 nm and λem = 535 nm) (Synergy H4 Hybrid reader (BioTEK, Winooski, VT, USA).

To evaluate ROS activity of C1, C2, and C3 in cellular models, the cell lines were plated in 96-well plates (5 × 103 cells/well) and 24 h later were treated with various compounds and with 5 μM DCF-DA for 4 min at 37 ◦C. After incubation, cells were washed with 1× PBS (phosphate buffered saline), pH 7.4 (VWR), and DCF-DA fluorescence was measured by using a multimode plate reader (λexc = 485 nm and λem = 535 nm) (Synergy H4 Hybrid reader (BioTEK)). Values were normalized on cell proliferation by an alamarBlue viability assay. Representative images of cells were obtained using a Leica DMI3000 M inverted microscope (Leica, Wetzlar, Germany) at 20× magnification. Images were analyzed using ImageJ software (NIH Image, Bethesda, MD, USA).

#### *2.4. Alamar Blue Viability Assay*

Cells were seeded in 96-well plates and the day after were incubated with various coordination compounds at the indicated conditions (see figure legends). At the end of the treatments, a stock solution of resazurin sodium salt (Sigma-Aldrich, St. Louis, MO, USA) (1 mg/mL) in PBS was diluted 1% (*v*/*v*) in complete DMEM or RPMI medium and added to the cells. After 3 h in the incubator (37 ◦C), the fluorescence was measured at 25 ◦C in a plate reader Synergy H4 Hybrid reader (BioTEK), λex = 550 nm, λem = 590 nm.

The fluorescent intensity measurements were processed using the following Equation:

% Cell viability = ((Sample data − Negative control)/(Positive control − Negative control)) × 100

The positive control corresponded with untreated cells. A resazurin solution without cells was used as a negative control.

#### *2.5. Western Blot Analysis*

Cells were harvested, washed in PBS, and re-suspended in RIPA buffer (Tebu-BIO #AR0105) in the presence of a protease inhibitor cocktail (Thermo Scientific™, Madrid, Spain #A32955). After incubation on ice for 30 min, the lysates were centrifuged at 14,000× *g* for 10 min at 4 ◦C and the supernatant fractions were used for Western blot analysis. Protein concentration was measured by Bradford reagent (Bio-Rad protein assay) using bovine serum albumin as a standard. Protein extracts (30 μg/lane) were resolved on a 10% SDSpolyacrylamide gel and electro-blotted onto PVDF membranes (AmershamTM ProtranTM 0.45 μm NC). Membranes were blocked in 5% low-fat milk in TBST or 5% BSA (50 mM Tris pH 7.5, 0.9% NaCl, 0.1% Tween 20) for 1 h at room temperature and probed overnight at 4 ◦C with a rabbit polyclonal anti-Bcl-2 (1:1000) (Cell Signaling, #2872), rabbit polyclonal anti-p70 S6 kinase (1:1000) (Cell Signaling, #9202), rabbit polyclonal anti-phospho-p70 S6 kinase (Ser371) (1:1000) (Cell Signaling, #9208), or mouse monoclonal anti-GAPDH (1:1000) (Santa Cruz, sc-47724). Horseradish peroxidase conjugated anti-mouse or antirabbit IgGs (1:5000 in blocking solution) (Santa Cruz, Spain) were used as secondary antibodies. Immunodetection was carried out using Bio-Rad chemiluminescent substrates and recorded using a Gel Documentation System (Syngene).

#### *2.6. Acquisition of NMR Spectra*

NMR spectra were recorded on a Bruker Advance 300 (1H: 400 MHz) spectrometer at 298 K using partially deuterated solvents as internal standards. Chemical shifts (δ) are denoted in ppm. Multiplicities are denoted as follows: s = singlet, d = doublet, t = triplet.

#### *2.7. Acquisition of FT-IR Spectra*

FT-IT spectra were recorded as neat samples in the range 400–4000 cm−1 on a Bruker Tensor 27 (ATR device) Spectrometer.

#### *2.8. Powder X-ray Diffraction Collection*

PXRD data were collected in a Rigaku Smartlab SE diffractometer with a Bragg-Brentano configuration, using Cu-Kα radiation (λ = 0.1541 nm). Samples were measured between 5◦ and 50◦ with a speed of 1.8◦ min−1 under an X-ray fluorescence reduction mode, at room temperature.

#### *2.9. Analysis of Synergy/Antagonism from Combination Studies*

To determine possible additive and synergistic effects when using combinations of **C-1**, **C-2** and **C-3** with GEM, the data from cell viability assays were first analyzed using the freely available software Combenefit [31], which simultaneously assesses synergy/antagonism using three published models (Highest single agent (HSA), Bliss, and Loewe).

Three concentrations were employed for **C-1**, **C-2**, and **C-3** (25, 500, and 1000 μM), while six concentrations were employed for GEM (0.5, 4.5, 20, 40, 60, 80 μM). All data were normalized to untreated controls, and imported into Combenefit software, where the Loewe Additivity model was employed to identify synergistic, additive, or antagonistic drug combinations. The software calculates the combination index (CI) for each drug combination, where a CI value < 1 indicates synergy, CI = 1 is additive and CI > 1 indicates antagonism.

#### *2.10. Annexin-V Assay*

Cells were seeded in 6-well plates and the day after were incubated with various coordination compounds at the indicated conditions (see figure legends). At the end of the treatments, the dead cells were collected and the attached ones were trypsinized and collected. The cells were washed with PBS 1× in suspension by centrifugation at 177× *g* for 5 min. Cells were resuspended in 100 μL binding buffer 1× and then 10 μL annexin V 1:10 was added and the cells were incubated for 15 min at 4 ◦C in darkness. After that, 380 μL binding buffer 1× were added to the samples and then 10 μL propidium iodide. The acquisition was performed in a Beckman Coulter Cytomics 500 Flow Cytometer using 20,000 cells in the Flow Cytometry Service at the CNB-CSIC.

#### *2.11. Statistical Analysis*

Comparisons among groups were analyzed via the independent-samples one-factor ANOVA test using Prism GraphPad software. All statistical data were obtained using a two-tailed student's *t*-test and homogeneity of variance tests (*p* values < 0.05 were considered significant).

#### **3. Results**

#### *3.1. Synthesis and Characterization of Three Iron-Based Trimers*

As mentioned previously, an elegant alternative to employ insoluble CPs in biological media is the use of the triazole-based trimer system [Fe3(NH2-trz)6]X6 and its derivatives [Fe3(RN-trz)6]X6. These systems are characterized by a backbone of three linearly arranged Fe(II) ions connected by three triazole ligands (Figure 1) [32–34]. The reduced length contributes to their solubility in contrast to that of the larger CPs. In addition, the steric hindrance of the ligands and the strong Fe-*N* coordination bond contribute to the stabilization of the bivalent iron under physiological conditions. The synthetic approach to obtain these water-soluble trinuclear systems resides in the use of p-toluenesulfonate (OTs) as counterions in highly diluted conditions to prevent or limit the formation of larger "Fe(II)/1,2,4-triazole" polymeric chains.

**Figure 1.** (**A**). Reaction required to obtain the trimers. (**B**). Representation of the different triazole derivatives employed (**C**). Schematic representation of the iron triazole-based trimers, where Fe is represented in orange, C in grey, N in blue, O in red, S in yellow, H in white, and the R group in black.

Three different Fe(II) trinuclear triazole-based CPs were synthesized in air conditions using ascorbic acid as the antioxidant, with general structure [Fe3(RN-trz)6(H2O)6], using iron(II) tosylate salt and triazole-based ligands: [(E)-*N*-(1-(thiophen-2-yl)ethylidene)-4H-1,2,4-triazol-4-amine (**C-1**), 4-amino-1,2,4-triazole (**C-2**), and (E)-*N*-(1-phenylethylidene)- 4H-1,2,4-triazol-4-amine] (**C-3**) (see Figures S1–S5). The stability of these in situ generated complexes in water and phosphate-buffered saline (PBS) was monitored through ESI-MS and specifically with UV-Vis spectra at the incubation conditions (i.e., at 10−5 mol·L−1, 37 ◦C and the spectra were taken at 0 h, 24 h, and 48 h) (Figures S6 and S7). It is noteworthy that the enhancement of the intensity of the band around 250 nm takes place without any shift, and also there is an absence of additional bands, which ratifies the stability of the CPs under these conditions. To further characterized these CPs, the solution was evaporated until precipitation and the resulting powders were characterized by FTIR and PXRD (See Figures S8–S11).

#### *3.2. Antitumoral Activity of Coordination Polymers*

Once the trimers were obtained, their effect on cell viability was evaluated in pancreatic cancer cell lines, based on previous reports where similar structures were employed in other tumor systems [29,30]. Intriguingly, after 48 h from the treatment, **C-1** and **C-2** were the most active in PANC-1, whereas **C-3** exerted a cytotoxic effect only in BXPC-3 cancer cells (Figure 2A,B). However, as illustrated in Figure S12, lower concentrations of trimers did not significantly affect cell viability.

Then, the different responses of pancreatic ductal adenocarcinoma cells to GEM were studied. First, as reported in previous studies [35], we observed that PANC-1 showed intrinsic chemoresistance to chemotherapy (Figure S13). To evaluate the potential effect as chemotherapy adjuvants of such derivates, the cell viability was studied when the trimers were combined with GEM. In the case of BXPC-3, the cell viability obtained in the presence of GEM did not change when it was combined with the trimers. Interestingly, in PANC-1, the combination of the trimers with GEM was able to overcome their intrinsic chemoresistance to this drug, especially when the chemotherapeutic was used at lower concentrations (0.5 and 4.5 μM) (Figure 2C and Figure S14). In particular, the combined treatment led to a synergistic enhancement of the cytotoxic effect, as shown by heat maps obtained with the Loewe Additivity model (Figure 2E) [31]. The synergistic effect is particularly relevant when the CPs were used at the concentration of 1000 μM and GEM at concentrations ranging from 0.5 to 20 μM. Then, to assess the specificity of these materials for cancer cells, similar experiments were also carried out in HaCaT cells, a non-cancerous cellular model. Interestingly, none of the three compounds exhibited toxicity in this cell line (Figure S15A) and did not enhance the cytotoxic activity of GEM (Figure S15B). These data suggest that these derivates are safe in non-tumor models and have therapeutic potential in the selective treatment of pancreatic cancer cells resistant to GEM.

**Figure 2.** Antitumoral activity of coordination polymers (CPs). (**A**,**B**) Cell viability studies in pancreatic cancer cell lines treated with **C-1, C-2**, and **C-3** for 48 h. (**C**,**D**) Cell viability studies in pancreatic cancer cell lines treated with 4.5 μM gemcitabine (GEM) and **C-1**, **C-2**, and **C-3** for 48 h. The values of treated cells were normalized to the untreated controls and reported as mean ± SEM. Statistical analysis was performed using one-way ANO VA (each group vs. control). (\*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05). (**E**) Combenefit-mapped surface output for the drug combinations involving **C-1**, **C-2**, and **C-3** with GEM in PANC-1 cells. The concentrations of each drug are plotted along the horizontal axes, while the percentages of cells remaining relative to untreated controls are plotted on the vertical axes. A heat map is used to represent the level of synergy (blue color) at each concentration. A heat map is used to represent the level of synergy (blue color) at each concentration. All experiments were conducted at least three times.

#### *3.3. The Combined Treatment Increased Apoptosis and Inhibited the mTOR Pathway in Pancreatic Cancer Cells*

To better understand the mechanism behind the antitumoral activity, some molecular markers related to apoptosis and cell growth in PANC-1 cancer cells were evaluated by Western blots (e.g., Bcl-2, p70S6K) and by Annexin-V assay. First, the effect of the

trimers in Bcl-2 was assessed. This protein contributes to cancer formation and progression by promoting the survival of cancer cells and represents a canonical target for cancer therapy [36]. Interestingly, CPs did not change the levels of Bcl-2 when administered alone (Figure S16), however, a reduction of Bcl-2 was observed when PANC-1 cells were treated with **C-1** and GEM. However, no significant reductions in this protein were observed after the treatment with **C-2** or **C-3** in combination with GEM (Figure 3A,B).

**Figure 3.** Apoptotic studies in pancreatic cancer cell lines. (**A**) Western blot analysis of Bcl-2 in PANC-1 cells after treatment with 1 mM **C-1, C-2, C-3**, and 4.5 μM GEM for 48 h. GAPDH protein level in the same extract was used as a control loading. (**B**) Densitometry of bands was performed using NIH Image J software and reported as fold change with respect to the untreated condition. The values reported are the mean of three independent experiments and were reported as mean ± SEM. (**C**) Cells were stained with Annexin V and propidium iodide (PI) and analyzed by flow cytometry. The percentage of cells in each group within the gated areas is indicated; the upper right panel represents cells undergoing late apoptosis, and the lower right panel represents cells undergoing early apoptosis. (**D**) Fold change in apoptosis. The values reported are the mean of two independent experiments and were reported as mean ±SEM.

Then, the cells were stained with fluorescein isothiocyanate (Annexin-V FITC) and propidium iodide (PI) and were analyzed by flow cytometry (Figure 3C,D). Interestingly, we observed an increase in Annexin-V fluorescence when the cells were treated with all three derivates and GEM, suggesting that the activation of apoptotic cell death may underlie the chemosensitivity observed.

Moreover, the activation of mTOR signaling after combined treatment was evaluated. It is worth mentioning that the inhibition in the mTOR signaling represents one of the mechanisms exploited by chemotherapy drugs to exert their antitumoral action [37]. Thus, when only GEM was administrated, chemoresistance in pancreatic cancer cells was induced. Remarkably, the administration of the therapeutic mixtures containing **C-1** or **C-2** or **C-3** and GEM, strongly abolished the chemoresistance mentioned before. Particularly, as discussed in below, they reduced the phosphorylation at Ser371 of p70S6 protein), indicating the downregulation of the mTOR pathway.

#### *3.4. Reactive Oxygen Species (ROS) Generated by Coordination Complexes*

Once the trimers were evaluated for their capabilities to synergistically increase the effect of GEM in PANC-1, the amount of ROS was evaluated [38], since they are usually generated in a variety of antitumoral systems. Thus, **C-1** to **C-3** were incubated with a standard ROS probe (DCFDA) at three different concentrations (1 μM, 100 nM, 1 nM) for 48 h. In this case, the fluorescent intensity increased in a concentration-dependent manner (Figure S17), highlighting the new trimers' potential use as ROS generators. Interestingly, the ROS production by addition of Fe(OTs)2 was significantly lower compared to the results obtained with CPs at the concentration of 100 nM (Figure S18). Then, the new trimers' activity was evaluated in two pancreatic cancer cell lines, PANC-1 and BXPC-3. Interestingly, **C-1** and **C-3** produced an enhancement on the ROS levels, whereas **C-2** did not provide any relevant effect compared to that of untreated samples (Figure 4A,B). This result suggests that **C-2** might be processed inside the cell differently, compared to **C-1** and **C-3**. Interestingly, the ROS production by addition of Fe(OTs)2 was significantly lower than in the case of **C-1** and **C-3** (Figure S19). These results suggest that iron-based CPs, such as **C-1** and **C-3**, can be employed to modify the oxidative status in pancreatic cancer cells, opening avenues to be exploited for therapeutic purposes.

Then, the activity of CPs was studied in combination with GEM, which is a standard agent used for the treatment of pancreatic cancer. It is important to point out that the cell lines (PANC-1 and BXPC-3) present different behavior against GEM. In particular, the presence of GEM induces a higher amount of ROS in BXPC-3 and reduces its viability more efficiently than in PANC-1 (Figure S20). In other words, as already mentioned above, PANC-1 presents chemoresistance to GEM, and for this reason, this cell line is often employed to assess novel therapeutics against pancreatic cancer. Thus, PANC-1 and BXPC-3 cancer cells were incubated with the three trimers and GEM for 48 h. In this case, a significant increase of ROS levels in both cell lines was observed when treated with **C-1** or **C-3** in the presence of GEM, compared to the ROS generated when treated only with GEM (Figure 4C,D). On the other hand, the combination **C-2** and GEM enhanced ROS only in PANC-1 cells. The ROS activity of the combined treatment was confirmed by fluorescent microscopy, where an increase in fluorescent signal was observed compared to that of the cells treated only with GEM (Figure 4E). Remarkably, when the iron salt and GEM were used, the ROS production was lower than in the cases where **C-1** and **C-3** were employed. This result highlights the advantage of the CPs evaluated herein compared to the iron salt Fe(OTs)2 (Figure S21). Then, similar experiments were also carried out in HaCaT cells. In this case, only **C-3** was able to increase ROS, and only weakly (Figure S22A). However, in the presence of GEM, a significant increase of ROS was observed in the case of **C-1** and **C-3**, as previously shown in pancreatic cancer cells (Figure S22B).

**Figure 4.** Reactive oxygen species (ROS) generated by coordination complexes. (**A**,**B**) ROS generation in pancreas cancer cell lines incubated with **C-1, C-2**, and **C-3** for 48 h. The DCF fluorescence (ROS production) of treated cells was normalized to untreated controls and reported as mean ±SEM. (**C**,**D**) ROS generation after combination therapy (incubation with GEM (4.5 μM) and **C-1, C-2**, and **C-3** for 48 h). The DCF fluorescence of treated cells was normalized to that of untreated controls and reported as mean ± SEM. Statistical analysis was performed using one-way ANOVA (each group vs. control). (\*\*\* *p* < 0.001, \* *p* < 0.05). (**E**) Fluorescence images of PANC-1 cells untreated and treated with the combined therapy. DCF, corresponding to ROS production levels are shown in green, and the nuclei are labeled in blue by Hoechst staining.

#### *3.5. Combined Treatment Attenuates the mTOR Pathway in an ROS-Dependent Manner*

Finally, the effect of ROS generated by the therapeutic mixtures in the regulation of cell proliferation was assessed by the addition of the standard inhibitor of oxidative stress, *N*-acetyl cysteine (NAC), which does not affect cell viability (Figure S23). In this case, the effect on cell viability by the therapeutic mixtures was attenuated after NAC administration in PANC-1 cancer cells (Figure 5A). Intriguingly, when the mixture contained **C-2** and GEM, the removal of ROS had no significant effect on cell viability, suggesting that its synergistic effect was independent of oxidative stress. Accordingly, the increasing of ROS after the combined therapy was reduced after NAC treatment (Figure S24A). Furthermore, the phosphorylation at Ser371 of p70S6K, was also dramatically recovered by the addition

of NAC (Figure 5B). These results highlight the role of ROS in the limitation of cancer cell growth mediated by the compounds described herein.

**Figure 5.** Combined treatment attenuates the mTOR pathway in an ROS-dependent manner. (**A**) PANC-1 cancer cell lines treated with 4.5 μM GEM, 500 μM NAC, and 1 mM **C-1** (blue), **C-2** (red), and **C-3** (green) for 48 h. Their viability was assessed with the alamarBlue test. The values of treated cells were normalized to that of untreated controls and reported as mean ± SEM. (**B**) Western blot analysis of phospho-p70SK6 and p70S6K of PANC-1 cells after treatment with 4.5 μM GEM, 500 μM NAC, and 1 mM of **C-1**, **C-2**, and **C-3** for 48 h. GAPDH protein level in the same extract was used as a control loading. (**C**) Densitometry of bands was performed using NIH Image J software and reported as fold change with respect to the untreated condition. The values reported are the mean of three experiments and were reported as mean ± SEM). Statistical analysis was performed using one-way ANOVA (C-1+GEM, C-2+GEM, C-3+GEM vs C-1+GEM+NAC, C-2+GEM+NAC, C-3+GEM+NAC)., \* *p* < 0.05).

#### **4. Discussion**

In this work, we used a particularly interesting family of iron CPs, which is the chainlike polymeric Fe(II) system, with general formula {[Fe(Rtrz)3]X2}n, (Rtrz = substituted triazole; X = monovalent anion), which has been largely studied in a solid state. These Fe(II) systems present the physical phenomenon of spin-crossover, which is accompanied by a strong thermochromism and a magnetic transition between diamagnetic and paramagnetic behavior in the solid state, either as a powder or as nanoparticles. These CPs have not been studied very much in the aqueous solution, mainly due to their poor solubility. To overcome this limitation, we used in this report shorter polymeric chains (more precisely, trinuclear oligomers), achieving unprecedented water-soluble derivatives (**C-1**, **C-2**, **C-3**).

Once these oligomers were synthesized and characterized by a multitude of techniques, their anticancer activities were studied for the first time. Remarkably, these derivatives were able to increase the activity of GEM in a pancreatic cancer cell line (PANC-1). Intriguingly, in BXPC-3, another pancreatic cancer cell line, the combined therapy did not enhance the chemotherapy effect, and the reduction observed was only due to GEM toxicity without any synergistic or additive effects. Furthermore, the specificity of our therapeutic systems was evaluated by assessing the activity of such derivates in HaCaT keratinocyte cells. In this case, none of the three compounds exhibited toxicity in this non-tumoral cell line and did not enhance the cytotoxic activity of GEM. These observations suggest that these derivates are safe in non-tumor models and have therapeutic potential in the selective treatment of pancreatic cancer cells resistant to GEM. The drastic difference between the pancreas cancer cell lines versus the non-tumoral model may be explained by, among other causes, acquired selective mutations, which lead to the regulation of different signaling pathways after GEM treatment. For example, chemoresistance of pancreatic cancer has been attributable to hyperactivation of MAPK [39], STAT-3, and NF-κB signaling pathways [40]. Moreover, the effects of GEM are mediated by transporters to cross extracellular or intracellular membranes, and oncogenic mutations occurring in solute carrier (SLC) and the ATP-binding cassette (ABC) superfamilies also have been linked to drug resistance in pancreatic cancer [41].

We investigated the molecular mechanisms underlying the activity of our CPs in pancreatic cancer cells. Interestingly, we observed that the CPs were able to attenuate chemoresistance in PANC-1 by inducing apoptosis and inhibiting mTOR signaling, which we detected through the phosphorylation of p70S6K, a classic downstream readout of this pathway. Curiously, although all three compounds were able to increase Annexin-v fluorescence when combined to GEM, the reduction of Bcl-2 protein, a classical inhibitor of apoptosis, was observed only when the therapeutic mixture contained C-1. This partial discrepancy could be explained in part because Bcl-2 s role in apoptosis is ambiguous. Indeed, Bcl-2 can also act as an apoptotic inducer blocking Bax/Bak oligomerization [42] and plays a role in other non-canonical functions including mitochondrial metabolism and biogenesis and autophagy regulation [43]. Another explanation could be that even if the translocation of phosphatidylserine is produced after the treatment, a process that is usually associated with the early stage of apoptosis, this does not result in the complete activation of the intrinsic apoptosis pathway and the formation of apoptosomes, which precedes caspase's activation [44].

ROS play a key role in various cellular processes, including proliferation, growth, apoptosis, and migration [45]. Importantly, ROS levels are frequently increased in cancer cells because of their high metabolic activity, mitochondrial dysfunction, and hyper-activation of oncogenes [46]. However, while ROS facilitate carcinogenesis and cancer progression with mild-to-moderate elevated levels, their excessive production may damage cancer cells dramatically through the modification of lipids, proteins, or DNA and lead to cell death [28]. Hence, ROS signaling and the antioxidant program play key roles in the response to chemotherapy and represent important targets to overcome drug-resistance [47].

In this study, the effect of our derivates on ROS metabolism in pancreatic cancer cells was investigated. Once we established that such structures were able to increase ROS production, both alone and in combination with GEM, we studied whether the cytotoxic effect of the combined therapy was due, at least in part, to the CPs' capacity to enhance ROS levels. To unveil this, experiments using the ROS scavenger NAC were performed, which showed that NAC could recover cell viability and the mTOR pathway affected by trimers and GEM.

Overall, these data allow us to speculate that both apoptosis induction and mTOR inhibition, resulting after combined therapy, might contribute to the synergistic reduction of cell viability observed in pancreatic cancer cells, and also that the cytotoxic effect is due to the increase in oxidative stress.

#### **5. Conclusions**

In summary, we have demonstrated that CPs can be used as adjuvants to enhance synergistically the activity of GEM in pancreatic cancer cell lines, which present intrinsic resistance to this chemotherapeutic through modulation of ROS levels. Interestingly, in contrast to that observed in PANC-1, the combined therapy did not enhance the chemotherapy effect either in BXPC-3, a pancreatic cancer cell line not resistant to GEM, nor in HaCaT keratinocyte cells, and the reduction of cell viability was only due to GEM toxicity without any synergistic or additive effect.

Furthermore, we studied the signaling pathways and molecular mechanisms underlying the chemosensitivity to GEM. We observed that the combined treatment increased apoptosis and reduced the phosphorylation of p70S6k, a well-established functional readout of the mTOR pathway involved in cell growth.

Finally, to unveil whether the increased ROS detected after combined therapy could have a role in the CPs' therapeutic effect, we performed functional studies using NAC, a standard inhibitor of oxidative stress. Notably, the addition of NAC significantly reverted the reduction of cell viability and the mTOR pathway observed after combined therapy, suggesting the CPs' effect was, at least in part, due to exacerbated ROS production in PANC-1 GEM-resistant cell lines.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-392 1/10/1/66/s1, Figure S1: Scheme of the reaction of the ligands TE-Tria and PE-Tria, Figure S2: 1H NMR (400 MHz, DMSO-d6, 298 K) of TE-Tria, Figure S3: Infrared spectrum of TE-Tria, Figure S4: 1H NMR (400 MHz, DMSO-d6, 298 K) of the PE-Tria, Figure S5: Infrared spectrum of PE-Tria, Figure S6: UV-vis spectra of (a) **C-1** in water, (b) **C-1** in PBS, (c) **C-2** in water, (d) **C-2** in PBS, (e) **C-3** in water, and (f) **C-3** in PBS, Figure S7: Solution of **C-1, C-2**, and **C-3** (10−2 M) in water initially and aged for 24 and 48 h (a) with ascorbic acid and (b) without ascorbic acid, Figure S8: Infrared spectrum of **C-1**, Figure S9: Infrared spectrum of **C-2**, Figure S10: Infrared spectrum of **C-3**, Figure S11: Powder X-ray Diffraction of **C-1**, **C-2**, and **C-3**, Figure S12: Cell viability studies in pancreas cells, Figure S13: Dose response curve of GEM, Figure S14: Dose response studies in pancreas cancer cells, Figure S15: Cell viability studies in human keratinocytes, Figure S16 CP administered alone does not affect Bcl-2 protein level, Figure S17: DCF fluorescence generated by coordination complexes, Figure S18: DCF fluorescence generated by salt and hydrogen peroxide, Figure S19: ROS generation in pancreas cancer cell lines, Figure S20: Different responses to gemcitabine in pancreatic cancer cells, Figure S21: ROS generation in pancreatic cancer cell lines after combined treatment, Figure S22: ROS generation in human keratinocytes, Figure S23: Cell viability studies in pancreas cancer cells, Figure S24: Combined treatment attenuates the mTOR pathway in an ROS-dependent manner.

**Author Contributions:** Conceptualization, M.C., J.S.C. and Á.S.; Data curation, M.C., P.M.-R. and E.R.-U.; Funding acquisition, J.S.C. and Á.S.; Investigation, M.C., P.M.-R. and E.R.-U.; Methodology, M.C., P.M.-R. and E.R.-U.; Resources, L.S. and A.B.; Validation, M.C., P.M.-R. and E.R.-U.; Writing original draft, M.C., J.S.C. and Á.S.; Writing—review and editing, M.C., E.R.-U., A.G., J.S.C. and Á.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2017-87305-R, CTQ2016-80635-P, RYC-2014-16866), Comunidad de Madrid (IND2017/IND-7809, PEJD-2017-PRE/IND-4037, P2018/NMT-4321), Asociación Española Contra el Cáncer, and IMDEA Nanociencia. IMDEA Nanociencia acknowledges support from the 'Severo Ochoa' Programme for Centres of Excellence in R&D (MINECO, Grant SEV-2016-0686).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article. The raw data of blots are available from the corresponding author upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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**Genetically Modified Mouse Mesenchymal Stem Cells Expressing Non-Structural Proteins of Hepatitis C Virus Induce E**ff**ective Immune Response**

**Olga V. Masalova 1,\*, Ekaterina I. Lesnova 1, Regina R. Klimova 1, Ekaterina D. Momotyuk 1, Vyacheslav V. Kozlov 1, Alla M. Ivanova 1, Olga V. Payushina 2, Nina N. Butorina 3, Natalia F. Zakirova 4, Alexander N. Narovlyansky 1, Alexander V. Pronin 1, Alexander V. Ivanov 4,\* and Alla A. Kushch 1**

Received: 5 December 2019; Accepted: 31 January 2020; Published: 2 February 2020

**Abstract:** Hepatitis C virus (HCV) is one of the major causes of chronic liver disease and leads to cirrhosis and hepatocarcinoma. Despite extensive research, there is still no vaccine against HCV. In order to induce an immune response in DBA/2J mice against HCV, we obtained modified mouse mesenchymal stem cells (mMSCs) simultaneously expressing five nonstructural HCV proteins (NS3-NS5B). The innate immune response to mMSCs was higher than to DNA immunization, with plasmid encoding the same proteins, and to naïve unmodified MSCs. mMSCs triggered strong phagocytic activity, enhanced lymphocyte proliferation, and production of type I and II interferons. The adaptive immune response to mMSCs was also more pronounced than in the case of DNA immunization, as exemplified by a fourfold stronger stimulation of lymphocyte proliferation in response to HCV, a 2.6-fold higher rate of biosynthesis, and a 30-fold higher rate of secretion of IFN-γ, as well as by a 40-fold stronger production of IgG2a antibodies to viral proteins. The immunostimulatory effect of mMSCs was associated with pronounced IL-6 secretion and reduction in the population of myeloid derived suppressor cells (MDSCs). Thus, this is the first example that suggests the feasibility of using mMSCs for the development of an effective anti-HCV vaccine.

**Keywords:** hepatitis C virus (HCV); mesenchymal stem cells (MSC); modified MSC; DNA immunization; nonstructural HCV proteins; immune response; HCV vaccine; myeloid derived suppressor cells (MDSCs)


*4.3. Impact of Variation on f(ζ)*

The effects of variation *β*1 and *β*2 on velocity distribution is shown in Figure 12. Figure 12 shows the solution plots for the velocity profile by varying of Prandtl fluid number and flexible number.

One may witness that: