Combining Magnetic Resonance Imaging with Photothermal Therapy

Apr 27, 2018 - The resulting product of CuS@BSA nanoparticles with their magnetic resonance imaging (MRI) and photothermal therapeutic capabilities co...
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Combining Magnetic Resonance Imaging with Photothermal Therapy of CuS@BSA Nanoparticles for Cancer Theranostics Zhongyun Chu,† Zhiming Wang,† Lina Chen,§ Xiaoshuang Wang,§ Chusen Huang,† Malin Cui,‡ Da-Peng Yang,*,‡ and Nengqin Jia*,† †

The Education Ministry Key Laboratory of Resource Chemistry, Shanghai Key Laboratory of Rare Earth Functional Materials, Department of Chemistry, Shanghai Normal University, Shanghai 200234, China ‡ College of Chemical Engineering and Materials Science, Quanzhou Normal University, Quanzhou 362000, China § Jingzhou Central Hospital, The Second Clinical Medical College, Yangtze University, Jingzhou 434020, China S Supporting Information *

ABSTRACT: The treatment of tumors has been a wide concern by a large number of scientific researchers. Combining magnetic resonance imaging (MRI) with photothermal therapy (PTT) enables cancer theranostics to be more efficient and accurate. Herein, we synthesized CuS@BSA nanoparticles with an average grain diameter of about 16.5 nm through a facile one-pot eco-friendly and user-friendly strategy and it was found to have strong near-infrared absorption property and magnetic resonance imaging ability so that it can be utilized as a multifunctional agent for cancer theranostic. The in vitro toxicity study showed that CuS@BSA nanoparticles with low toxicity could kill cancer cells with the 980 nm NIR laser effectively. Furthermore, they exhibit a certain value of relaxivity (r1 = 0.26 mM−1·s−1) compared to that of clinically widely used reagent Magnevist (r1 = 3.13 mM−1·s−1). The resulting product of CuS@ BSA nanoparticles with their magnetic resonance imaging (MRI) and photothermal therapeutic capabilities could represent a kind of potential candidate for cancer theranostics. KEYWORDS: CuS@BSA nanoparticles, theranostics, low cytotoxicity, photothermal therapy (PTT), MRI contrast agent

1. INTRODUCTION As we all know, tumor has become a serious threat to human health; even worse, it has been the chief factor of mortality worldwide for decades.1 It is gratifying that after great efforts, diverse treatments have been developed to fight cancer, such as radiotherapy and chemotherapy.2,3 At the same time, people have also recognized the side effects on human health that cannot be ignored. Among various strategies, photothermal therapy (PTT) has drawn great attention recently and can be seen as a great development alternative or complement to traditional cancer therapies.4−6 The development of molecular imaging technology provides a new idea for modern medicine. As a common but predominant molecular imaging method in medicine, MRI has constantly been employed in the diagnosis of different diseases; the reason is that for ductile tissues it has excellent spatial resolution.7−11 PTT normally relies on the principle of light−heat conversion of the material with light absorption ability.12,13 Ideal PTT agents are supposed to exhibit salient absorbance in the near- infrared region (NIR). On the basis of the principle of photothermal conversion, it will convert into energy after absorbing light14,15 to achieve the purpose of tumor treatment by means of tumor ablation. Researchers have recently made great efforts to investigate the therapeutic effects of PTT in © XXXX American Chemical Society

numerous animal experiments, employing many kinds of nanomaterials as PTT agents.16−22 Vascular parts of the tumor with irregular tissue characteristics, resulting in tumor tissue acidic and hypoxic characteristics, are more sensitive to temperature compared with the normal tissue.23,24 When the temperature reaches 40−45 °C, tumor cells will undergo a series of changes including mitochondrial swelling, protein denaturation, and cell membrane rupture.25,26 These abnormal changes eventually cause tumor cell apoptosis, whereas no significant change is observed from normal tissue cells under the same experimental conditions for 1 h. Therefore, PTT has the capability to achieve targeted treatment of cancer cells. At the same time, PTT will not cause systemic reactions, with the advantage of low side effects. When it comes to disadvantage, there is no doubt that the penetration of PTT is the main factor restricting its development. Today, nanotechnology is developing at a high speed; in the near-infrared region, nanomaterials with good NIR absorption and high light-to-heat conversion efficiency have shown great promise in PTT applications Received: March 14, 2018 Accepted: April 27, 2018 Published: April 27, 2018 A

DOI: 10.1021/acsanm.8b00410 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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2. MATERIALS AND METHODS

To date, multiple nanomaterials with infrared absorption such as nanoparticles,27 carbon-based nanostructures, organic dyes, semiconducting compounds, noble metals, and polymers have been served as PTT agents.28−34 Typically, copper sulfide (CuS) attracted wide attention due to its low toxicity, facile preparation, low cost, and superior stability.35−42 In addition, it is generally believed that the mechanism of infrared absorption of CuS nanoparticles is due to the d−d transition of copper ions; therefore, CuS nanoparticles are able to be applied to PTT. Yet CuS nanoparticles have already been synthesized in some organic systems or simple aqueous solutions, and many ligands such as sodium citrate and sodium dodecyl sulfate, etc. have been exploited to synthesize CuS nanoparticles.43−48 However, it is worth noting that the lack of toxicity of CuS nanoparticles greatly limits its further application. Consequently, it is of vital importance to develop a simple, green, lowcytotoxicity, and cost-saving strategy to receive CuS nanoparticles for PTT. On the basis of the above, a simple strategy was proposed to mediate the synthesis of CuS nanoparticles using BSA as a biological template in this work, and then the prepared nanoparticles were applied to the photothermal treatment. As can be seen from the flowchart of this experiment (Scheme 1),

2.1. Materials and Reagents. All analytically pure reagents used in this work were used as originally. The reagent of cell counting kit-8 (CCK-8) was provided by Dojindo Laboratory. Trypan blue, propidium iodide (PI), and Hoechst 33258 were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). The lung adenocarcinoma cells (A549) and liver normal cells (LO2) were acquired from the cell bank of the Chinese Academy of Science (Shanghai, China). All cell culture reagents including fetal bovine serum (FBS) and medium (RPMI-1640) were received from Gibco. Lyophilized and powdered bovine serum albumin (BSA, 99%) was acquired from Sigma-Aldrich (USA). Fluorescein isothiocyanate (FITC) was obtained from Energy Chemical (Shanghai, China). Thioacetamide (TAA), copper nitrate (Cu(NO3)2), and nitric acid (HNO3) were purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Regenerated cellulose membrane (MWCO = 7000) was obtained from Yuanye Biotechnology Co., Ltd. (Shanghai, China). All aqueous solutions were obtained with Ultrapure water. 2.2. Apparatus. Conventional measurements of transmission electron microscopy (TEM) were performed via a JEOL JEM-2100 microscope (acceleration voltage remains 200 kV). Malvern Zetasizer Nano ZS90 was used to understand the ζ potential value and hydrodynamic size. UV−visible spectra were calculated utilizing a Varian Cary-Eclipse 500 spectrophotometer. The electronic states and element composition were measured by X-ray photoelectron spectroscopy (XPS, PerkinElmer PHI 5000C ESCA). Fourier transform infrared (FTIR) spectra was acquired by a Nicolet 380 spectrometer. A NIR laser (980 nm) was emitted by semiconductor exciter subsystem (BWT Beijing LTD). 2.3. Synthesis of CuS@BSA Nanoparticles. In our work, CuS@ BSA nanoparticles were prepared via a simple one-pot synthesis route in aqueous solution using BSA as biological template, Cu(NO3)2 as copper source, TAA as sulfur source at room temperature, and the ratio of Cu(NO3)2/BSA remains at 130:1. The first part, an aqueous Cu(NO3)2 (10 mM, 5 mL) and BSA (5 mg/mL, 5 mL) solution were mixed in the container of beaker with stirring for 30 min. Upon being mixed, a HNO3 solution (65 wt %) was added into the system to adjust the pH (pH = 3), and then a TAA solution (200 mM, 0.5 mL) was injected and incubated in water bath (90 °C) for 1 h. On the basis of the above basis, the raw product was centrifugated (10 000 rpm, 30 min). Then excess salt precursor was removed via a sufficient dialysis. The samples were obtained by lyophilization and dissolved in phosphate buffer solution (PBS) for the next experimental study. 2.4. Photothermal Ability of CuS@BSA Nanoparticles. In this work, 980 nm laser was used for next experiments, the temperature was recorded per 30 s throughout this part. The product of CuS@BSA solution (2 mL) at various concentrations (Cu2+ = 6.25,12.5, 25, 50 ppm) was put into a clean quartz cuvette, and it was illuminated under the laser for 5 min. Simultaneously, the PBS group was set as a control. Furthermore, the effect of a series of power density (0.79, 1.57, 2.79 W/cm2) on CuS@BSA was measured. After the irradiation, the laser was removed and the temperature of CuS@BSA nanoparticles (50 ppm) gradually decreased to room temperature. All the experiments were performed five times. Moreover, photothermal stability of nanoparticles was studied as presented in UV−vis spectra as well as size distribution before and after irradiation. 2.5. Cell Culture and in Vitro Cytotoxicity Assay. The cytotoxicity analysis of CuS@BSA was measured using CCK-8 assay on A549 and LO2 cells. They were cultured in cell incubator with a suitable humidified atmosphere (37 °C, 5% CO2), and RPMI-1640 medium which contains 10% FBS was provided. Typically, A549 cells (groups 1, 2) and LO2 cells (groups 3, 4) were seeded into four 96well plates. All of them were cultured in the same cultural environment for 1 day. After the incubation, all of the liquid was removed separately. Soon afterward they were feed with fresh complete culture medium (100 μL) (Cu2+ = 0, 6.25, 12.5, 25, 50 ppm) for 1 day (groups 1, 3) and 2 days (groups 2, 4). After that, CCK-8 was injected into the wells after the pipet of medium and the plate went on an

Scheme 1. Graphic Solution of Fabrication of CuS@BSA Nanoparticles for the Applications of PTT and MRI

the formed CuS@BSA nanoparticles have a relatively narrower size distribution with a mean particle diameter of 16.5 ± 2.2 nm. Furthermore, the experimental results showed that CuS@ BSA nanoparticles exhibit low cytotoxicity and excellent photothermal stability. In addition, in vitro experiments showed that CuS@BSA nanoparticles displayed an effective killing ability to A549 tumor cells under NIR laser of 980 nm. Moreover, further studies showed that it could act as a potential T1 MRI contrast agent. In conclusion, as an interesting kind of reagent with the property of photothermal effect, the nanoparticles we have fabricated possess many advantages such as ideal light-to-heat conversion efficiency, low cytotoxicity, and select photothermal stability which may be promising in the future field of tumor treatment. B

DOI: 10.1021/acsanm.8b00410 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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Figure 1. Characterization of CuS@BSA nanoparticles: representative images of TEM (a), EDS analysis (b), hydrodynamic size (c), and ζ potential (d).

Figure 2. Characterization of CuS@BSA nanoparticles: representative images of XPS (a) and FTIR spectra (b). washing with PBS, fluorescence microscope was run to capture cells with the excitation of the ultraviolet and blue channels, respectively. 2.7. In Vitro Photothermal Therapy of CuS@BSA Nanoparticles. As with the previous treatment, A549 cells were first incubated in plates (96 wells, groups 1, 2) for 24 h prior to a treatment with CuS@BSA at sundry concentrations (Cu2+ = 0, 6.25, 12.5, 25, 50 ppm) for 1 day. Hereafter, they (group 1) were irradiated by the laser (1.57 W/cm2) for 5 min, and the viability was measured. Photothermal effect was investigated with the help of trypan blue and PI. Briefly, A549 cells divided into different groups (groups 1−4) were inoculated into a plate. Then, they were treated with culture medium only, the laser (1.57 W/cm2, 5 min) only, CuS@BSA (Cu2+ = 25 ppm), and CuS@BSA (Cu2+ = 25 ppm) with the laser (1.57 W/ cm2, 5 min), respectively. Trypan blue solution was then transferred into each well. Similarly, A549 cells stained by PI underwent the same treatment. 2.8. T1 MRI Relaxometry Measurement. Both the longitudinal (T1) relaxometry measurements and the MRI were carried out via a previous reported method. And the experimental parameters are the same.49

incubation for further 2 h. At last, the absorbance value for each well at 450 nm was recorded. All of the results in this part were shown with error bars. 2.6. Cellular Uptake of CuS@BSA Nanoparticles. Distribution of CuS@BSA nanoparticles in the cells was explored through optical cellular observations, biotransmission electron microscopy, and fluorescence localization analysis. In a typical process, A549 cells were first incubated for 1 day before an incubation with CuS@BSA nanoparticles (Cu2+ = 25 ppm). Subsequently, they were washed by PBS, then gathered and fixed with 2.5% glutaraldehyde (diluted with PBS) overnight (4 °C). And then cells were fixed with 2% osmium tetroxide (OsO4, diluted with PBS) for 2 h (4 °C). After PBS washing, the cell sample was dehydrated with 30%, 50%, 70%, 80%, 90%, and 100% ethanol gradually. Afterward, it was moved into Epon resin and polymerized to prepare ultrathin sections with a thickness of 60 nm. Then this sample was placed on a copper mesh, and then uranyl acetate and lead citrate were used to stain it for 5 min, respectively. Finally, morphology of cells was observed via biotransmission electron microscopy. Correspondingly, cells not incubated with CuS@BSA nanoparticles were set as the negative group. For fluorescence localization analysis, CuS@BSA nanoparticles were first mixed with FITC solution in aqueous solution with a stir for 24 h, followed by a centrifugation and sufficient dialysis. Afterward, A549 cells were incubated in cell incubator with the product CuS@BSAFITC (Cu2+ = 25 ppm) for 6 h. At the same time, the nucleuses of A549 cells for cell labeling were stained by Hoechst 33258. After

3. RESULTS AND DISCUSSION 3.1. Characterization of CuS@BSA Nanoparticles. BSA, a common commercially available protein that acts as a stabilizing agent, has a multiplicity of amino acid residues and C

DOI: 10.1021/acsanm.8b00410 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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the peaks emerging at 1384 cm−1 and 1084 cm−1 could be caused by several factors such as bending vibration, methylene volatility, and primary amine C−N telescopic. Furthermore, the phase of CuS@BSA nanoparticles was studied. As displayed in Figure 3, the XRD picture suggests that CuS@BSA nano-

sulfhydryl bonds.50 What is interesting is that they can combine with metal ions to form a complex by continuous stirring when the metal ions are added into the system.51 Moreover, BSA with unique structure has been successively developed as a natural biotemplate in the process of biomineralization for great variety of nanomaterials. Accordingly, in this work, BSA was naturally selected as a templating material, Cu(NO3)2 was treated as the origin of copper, and the element of sulfur was from TAA. Under continuous stirring, Cu(NO3)2 and BSA formed a stable solution. What is even more interesting is that when we control the ratio of Cu(NO3)2 and BSA at 130:1, the resulting nanoparticles have better water solubility and stability. At the same time, we are also considering the effect of the subsequent application of Cu(NO3)2 and BSA ratios in the product, which requires further study. It is worth noting that BSA with excellent low toxicity and abundant functional groups on its surface plays a great role in the synthesis of CuS@BSA nanoparticles. The structure, morphology, and size were characterized and shown in Figure 1 and Figure 2. As shown in TEM image (Figure 1a), CuS@BSA nanoparticles were dispersed evenly without aggregation, and it has narrow distribution with a diameter of 16.5 ± 2.2 nm as shown in the inset of Figure 1a, which was calculated on the basis of the measurements of 200 nanoparticles arbitrarily. Additionally, as shown in Figure 1c, the mean hydrodynamic diameter is about 37 nm (PDI = 0.315), which shows some difference with the mean diameter from TEM. The reason for this difference is that the TEM measures the size of the individual CuS@BSA nanoparticle, while DLS may measure its hydrodynamic size.52,53 In future applications, this kind of nanomaterial with such particle size may produce EPR effects in specific cancerous tissues, which is very beneficial for the theranostics of tumor. Besides, asprepared CuS@BSA nanoparticles have colloidal stability with the unique ζ potential of +37.4 mV (Figure 1d), which can easily target-bind negatively charged tumor cells.54,55 The EDS (Figure 1b) result shows the presence of several kinds of elements such as copper, nitrogen, sulfur, and oxygen. It must be noted that the element of silicon and molybdenum could be attributed to the formvar stabilized with carbon support films (Mo). In addition, XPS (Figure 2a) was performed to characterize the chemical components and electronic state on the surface of CuS@BSA. Figure 2a shows the result of the XPS spectrum. In the spectrum, the peaks corresponding to Cu 2p, C 1s, N 1s, O 1s, and S 2p can be obviously identified, suggesting the presence of both Cu and BSA in as-prepared CuS@BSA nanoparticles. Additionally, the percentage of each element was clearly presented in the inset of Figure 2a. As presented in the inset, the atomic ratio of Cu/S was about 0.424:1, which is less than that calculated in the reactants of TAA and Cu(NO3)2. The reason is that BSA contains sulfur element. Moreover, the high resolution XPS spectra of Cu 2p optimized by the Lorentzian−Gaussian method can be seen in Figure 2a. The peaks at ∼933.3 eV and ∼953.7 eV are respectively from Cu 2p3/2 and Cu 2p1/2. Accordingly the peaks at ∼941.1 eV and ∼963.3 eV are characteristic peaks of Cu2+ (3d9). All of the results reveal that copper is present in the form of Cu2+, and the composition of the sample is CuS. At the same time, FTIR spectroscopy was carried out to characterize synthetic nanoparticles. As shown in Figure 2b, it clearly displays the typical peaks of BSA. The peak at 3443 cm−1 can be attributed to O−H, and 2960 and 1651 cm−1 are due to amide A and amide I, respectively. Moreover,

Figure 3. XRD analysis of CuS@BSA and JCPDS card no. 06-0464.

particles own a certain degree of crystallinity and blatant diffraction peaks. The main peaks at 29.27°, 31.78°, 32.85°, 47.94°, 52.71°, and 59.34° are corresponding to the planes of (102), (103), (006), (110), (108), and (116), respectively. All of the above results were able to certificate the integral synthesis of CuS@BSA nanoparticles. 3.2. Stability of CuS@BSA Nanoparticles. The stability in different acid solutions or diverse solution system of nanomaterial is the condition that it can be employed in biomedical applications; hence this property of CuS@BSA must be investigated. In this study, discriminate acid buffer solution (pH = 4.0−7.4), saline, water, RPMI-1640, and FBS were prepared to dilute CuS@BSA nanoparticles at room temperature. The concentration of copper was constant at 25 ppm. One month later, no visible aggregation could be found, revealing ideal colloidal stability of CuS@BSA nanoparticles. Then, UV−visible spectrometry in the range of 400−1000 nm was used to the record the absorbance values. By the way, the absorbance at 980 nm was obtained. As depicted in Figure 4a, there is no any apparent value change in absorption features of CuS@BSA nanoparticles after incubation with all of the above solutions. Obviously, the absorbance at 980 nm (Figure 4b) of the nanoparticles does not change significantly in the mentioned solution, suggesting that the nanoparticle is stable. Thus, taking the results into consideration, it is not difficult to get the fact that the nanomaterial has a delightful stability, and it paves the way for the next application. 3.3. Photothermal Effect of CuS@BSA Nanoparticles. Inspired by the obvious absorbance in the NIR region, photothermal property was further investigated. In this study, the absorbance of CuS@BSA nanoparticle dissolved in PBS at different concentrations (Cu2+ = 6.25, 12.5, 25, 50 ppm) was studied with the UV−visible spectrometer; simultaneously, the absorbance at 980 nm was recorded. Obviously, the result (Figure 5a) shows that CuS@BSA nanoparticles have a good absorption effect in the NIR window. Moreover, the linear coefficient of 0.999 94 shows that the nanomaterial concentration was positively correlated with the absorbance at 980 nm as displayed in Figure 5c. Naturally, the laser at 980 nm was exploited in the experiments. Additionally, Figure 4b presented that the temperature of nanomaterial at various concentrations changed significantly with the laser (1.57 W/cm2, 5 min), while D

DOI: 10.1021/acsanm.8b00410 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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Figure 4. UV−vis spectra (a) of CuS@BSA in the pH = 4.0−7.4 buffer, saline, water, RPMI-1640, and FBS and the corresponding absorbance of CuS@BSA at 980 nm (b).

Figure 5. UV−vis spectra (a), the temperature (b), the absorbance at 980 nm (c) of CuS@BSA at diverse concentrations of Cu. The temperature separation of CuS@BSA (50 ppm) and PBS under laser with diverse power densities for 5 min (d). The temperature fluctuation of CuS@BSA after laser (1.57 W/cm2, 5 min) (e) and an interval of 10 min after 5 cycles.The UV−vis spectra before and after irradiation (1.57 W/cm2, 30 min) (f).

there is no distinctive change that can be seen at 0 ppm. As presented, the concentration at 50 ppm has the most significant change. Accordingly this concentration was chosen to monitor the photothermal effect. After irradiation (1.57 W/cm2, 5 min), the nanoparticles at 50 ppm emerged with a palpable temperature increase ∼40 °C (Figure 5d) compared to PBS. To our knowledge, photothermal stability is of vital importance to the application; hence photothermal stability of CuS@BSA nanoparticles has to be taken into consideration. With the laser (1.57 W/cm2, 5 min), the temperature of nanoparticles increased rapidly, and then the NIR irradiation was removed. In this process, the temperature was recorded until room temperature. This operation was repeated five times. Figure 5e shows that the temperature curve of CuS@BSA performs a similar trend, indicating that the photothermal conversion efficiency is always ideal and does not change. On the basis of the UV−vis spectra as shown in Figure 5b and Figure 5f and TEM observation (Figure S1), there was no obvious value and morphology change. Due to these properties, CuS@BSA

nanoparticles are capable of exhibiting good photothermal stability in this study. 3.4. Cytotoxicity Analysis of CuS@BSA Nanoparticles. Cytotoxicity of CuS@BSA nanoparticles at different concentrations was evaluated on A549 cells and LO2 cells via CCK-8 method after incubation. As seen in Figure 6a, the viability of

Figure 6. CCK-8 assay of A549 (a) and LO2 (b) cells viability treated by CuS@BSA (6.25−50 ppm). E

DOI: 10.1021/acsanm.8b00410 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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Figure 7. Fluorescent signal images of A549 cells incubated with Cu@BSA-FITC (25 ppm) for 6 h. The scale bars in the images (a−c) are 50 μm. TEM images of A549 cells: negative control cells without treatment (d) as a comparison, cells after incubation with Cu@BSA (25 ppm) for 12h (e), and a local enlarged view (f) of the highlight area in (e).

A549 cells still remains more than 80% after incubation for 1 day and 2 days, while Figure 6b shows that it remains at least 90% for LO2 cells, indicating that CuS@BSA nanoparticles express the properties of low cytotoxicity. 3.5. In Vitro Cellular Uptake of CuS@BSA Nanoparticles. To investigate whether CuS@BSA nanoparticles can be internalized by tumor cells, A549 cells were selected as the model to evaluate their cellular uptake. In this study, biological TEM and fluorescence photography were used to track the internalization of CuS@BSA nanoparticles. In the first place, A549 cells were incubated with CuS@BSA-FITC (Cu2+ = 25 ppm), and then their nucleuses were stained by the dye Hoechst 33258. Figure 7a−c shows the photographs of A549 cells treated by CuS@BSA-FITC for 6 h. It is clear that obvious fluorescent signals can be obtained around the nucleus owing to an efficient uptake of the nanoparticles, indicating that CuS@ BSA-FITC nanoparticles are endocytozed into the cytoplasm of A549 cells. To further certificate the subcellular level distribution of CuS@BSA nanoparticles, biological TEM was also carried out to observe the dispersion of them. Chromatic aberration in bright and dim areas can be observed from Figure 7e, which indicates that they are distributed in the cytoplasm too. Figure 7f is a partially enlarged view of the area which is marked in Figure 7e. It is obvious that nanoparticles are captured by lysosome, while they are not able to be seen in the control (Figure 7d). All of the phenomena show that CuS@ BSA nanoparticles can be internalized by cells. 3.6. CuS@BSA Nanoparticles for in Vitro PTT. A549 cells treated with CuS@BSA were divided into two groups (control and experimental), and then A549 cells at the experimental groups were irradiated using the laser wavelength at 980 nm for 5 min. CCK-8 method was performed to appraise the cell viability. From Figure 8, it is obvious that with the increase of concentration of CuS@BSA, obtained cell viability decreases gradually. When it reached 50 ppm, the cell survival rate of experimental groups remained only 17.5%, while the

Figure 8. CCK-8 based assay of A549 cells after incubation with CuS@BSA for 24 h without (black) and with laser irradiation (red, power density of 1.57 W/cm2, 5 min).

control group cell survival rate was as high as 85%, indicating that as-prepared CuS@BSA nanoparticles have a powerful ability to kill tumor cells. In order to study the PTT effect on the cells, trypan blue and PI were performed to stain cells, respectively. As shown in the trypan blue stained fluorescence images (Figure 9a−d), A549 cells in blank group (a), A549 cells with laser for 5 min (b), and A549 cells with CuS@BSA nanoparticles (Cu2+ = 25 ppm) for 24 h, only (c) showed no significant fluorescence signal, while A549 cells incubated with CuS@BSA nanoparticles (25 ppm) for 24 h and laser irradiation emitted strong red fluorescence signals (d). Further, the treatment of A549 cells stained by PI was the same as that of the cells stained by trypan blue. Interestingly, A549 cells incubated with CuS@BSA nanoparticles (Cu2+ = 25 ppm) for 24 h and laser irradiation emitted strong blue fluorescence signals (h), while the other three groups did not show any similar fluorescence signals. These phenomena further prove the PTT effect of CuS@BSA nanoparticles. 3.7. In Vitro MRI Performance. MRI plays an important role in molecular imaging technology, which is currently widely F

DOI: 10.1021/acsanm.8b00410 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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Figure 9. Optical images stained by trypan blue (a−d) and fluorescence images stained by PI (e−g): A549 cells without treatment (a, e); A549 cells treated with laser for 5 min (b, f); A549 cells incubated with CuS@BSA for 24 h without laser irradiation (c, g). A549 cells treated with CuS@BSA nanoparticles for 24 h under laser irradiation for 5 min (d, h).

assay. In vitro experimental results presented in this work reveal the significant capability to kill tumor cells under 980 nm NIR laser. As-prepared nanoparticles with a mean size of 16.5 nm exhibit a certain potential for T1-weighted MRI performance, efficient NIR photothermal conversion ability, ideal distribution, and photothermal stability. In other words, CuS@BSA nanoparticles have an enlightening influence on future photothermal therapy applications.

applied in clinical practice. To study the MRI effect of CuS@ BSA nanoparticles as an efficacious T1 contrast agent, the relaxation time was calculated. As shown in Figure 10a, MRI

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b00410. TEM images of CuS@BSA before and after the laser irradiation (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*D.-P.Y.: e-mail, [email protected]. *N.J.: e-mail, [email protected]. Figure 10. MRI images of CuS@BSA at different concentrations (a) and trend of 1/T1 and Cu concentration in CuS@BSA aqueous solution (b).

ORCID

images of CuS@BSA nanoparticles at different concentrations were obtained, indicating that the brightness increases with the CuS@BSA nanoparticles concentration. Similarly Figure 10b shows a regular and improved signal with the change of concentration. At the same time it shows r1= 0.26 mM−1·s−1, which is lower than Magnevist (r1 = 3.13 M−1·s−1). Of course this provides a new idea for the next development of MRI contrast agent in medical imaging.

Notes

Chusen Huang: 0000-0002-2481-9147 Da-Peng Yang: 0000-0003-3509-2825 Nengqin Jia: 0000-0003-0761-8877 The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We sincerely appreciate the support from Shanghai Science and Technology Committee (Grant 17070503000), National Natural Science Foundation of China (Grants 21373138 and 81472001), Program for Changjiang Scholars and Innovative Research Team in University (Grant IRT_16R49), and Science and Technology Innovation Foundation for College Students from Shanghai Normal University.

4. CONCLUSIONS In summary, in this work, BSA served as a biotemplate with good water solubility to synthesize CuS@BSA nanoparticles via a facile strategy; this one pot method in this work was found to be eco-friendly and highly effective. As a result of the green strategy, no visible toxicity was observed in vitro via CCK-8

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REFERENCES

(1) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. Global Cancer Statistics. Ca-Cancer J. Clin. 2011, 61, 69−90.

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DOI: 10.1021/acsanm.8b00410 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX