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Albumin Assisted Synthesis of Ultra-small FeS2 Nanodots for Imaging-guided Photothermal Enhanced Photodynamic Therapy Qiutong Jin, Jingjing Liu, Wenjun Zhu, Ziliang Dong, Zhuang Liu, and Liang Cheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16890 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 10, 2017

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ACS Applied Materials & Interfaces

Albumin Assisted Synthesis of Ultra-small FeS2 Nanodots for Imaging-guided Photothermal Enhanced Photodynamic Therapy Qiutong Jin, Jingjing Liu, Wenjun Zhu, Ziliang Dong, Zhuang Liu, Liang Cheng* Institute of Functional Nano & Soft Materials Laboratory (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou, Jiangsu 215123, China E-mail: [email protected]

Abstract: Current mainstream cancer treatment methods have their limitations. New approaches are thus desired to assist our battle against cancer. Herein, multifunctional ultra-small FeS2 nanodots with the size of 7 nm are synthesized by biomineralization and used for imaging-guided combined tumor therapy. Bovine serum albumin (BSA) which acts as the reaction template to induce the mineralization of FeS2 nanomaterials under alkaline condition, could also be used as a drug delivery system for coupling photosensitive molecule such as chlorin e6 (Ce6). Taking advantages of the near-infrared (NIR) absorbance and the high r2 relaxivity of the synthesized ultra-small FeS2 nanodots, as well as the Ce6 fluorescence, in vivo tri-modal imaging of optical / magnetic resonance / photoacoustic was carried out, showing efficient tumor accumulating of FeS2@BSA-Ce6 after intravenous injection. In vitro and in vivo photothermal & photodynamic therapy was then conducted to get synergistic tumor therapy and did not cause any apparent toxicity to the treated animals. Our

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work thus provides a new kind of ultra-small FeS2 multifunctional nanodots modified by albumin via a simple method, promising for combination phototherapy as well as cancer theranostics.

Keywords: Albumin; ultra-small FeS2 nanodots; multimodal imaging; phototherapy; combined therapy

1. Introduction Cancer is one of the major diseases threatening human life and health.1, 2 Commonly used traditional clinical cancer therapies, including surgery, chemotherapy, and radiotherapy, all have their own limitations such as severe side effects and drug resistance.3-5 Therefore, it has become one of the hot issues to effectively improve the existing treatment methods for cancer therapy, such as tumor-targeted delivery and imaging-guided cancer therapy.5-7 Various kinds of nanomaterials, which have interesting optical, magnetic, acoustic, or other unique physical properties, could be utilized to develop new types of biomedical imaging technologies, which would facilitate early detection of cancer and enable better therapeutic planning.8-14 Moreover, many functional nanomaterials could even be utilized to develop new types of treatment strategies such as phototherapy,8 magnetocaloric therapy,15 and immunotherapy16, 17 to achieve safe and efficient cancer treatment. Phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), has recently attracted widespread interests for the reason that it could be easily


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triggered by light as a minimal-invasive therapeutic approach.8 Under the light irradiation, photo-absorbing nano-agents in PTT could transfer light energy into heat, which leads to the hyperthermia in tumors. On the other hand, tumor-targeting nanomaterials could carry photosensitizing (PS) molecules for PDT to kill nearby cancer cells by generating toxic reactive oxygen species (ROS). Moreover, the two categories of phototherapies could work together to kill cancer in a synergistic manner.18,

19

Nowadays, a wide range of PS nanocarriers have shown excellent

outcomes both in vitro and in vivo,20-24 attracting increasing interests in the nanomedicine field. Iron-based functional nanomaterials, mainly including iron oxides and iron sulfides, have been widely used in nanomedicine.25-27 Various kinds of multifunctional superparamagnetic iron oxide (Fe3O4) nanostructures have been synthesized and used for magnetic resonance (MR) imaging-guided phototherapy of cancer.8, 26 In addition to their MR contrasting abilities, iron sulfide nanomaterials would often exhibit much stronger NIR absorbance compared to the iron oxide, and would be promising for PA / MR imaging-guided cancer therapy.28-30 However, many previously reported FeS2 nanomaterials have relatively large sizes and are difficult to be excreted, leading to the concerns about their potential toxicity.31 Considering the unique structure of the glomerular capillary wall, ultra-small nanoparticles can pass through the kidneys and undergo rapid renal excretion.32, 33 Therefore, it would be interesting to develop safe and functional ultra-small FeS2 nanoparticles for theranostic applications.



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Biomineralization is the process by which organisms control the formation of inorganic minerals through the regulation of biological macromolecules.34, 35 In recent years, the synthesis of functional nanomaterials by protein biomineralization has also been reported, such as CuS, Bi2S3, and WO3 ultra-small nanodots.36-38 Herein, we used a protein-templated biomineralization method to synthesize ultra-small biocompatible FeS2 nanodots with unique optical and magnetic properties. After the conjugation with chlorin e6 (Ce6), a typical PS molecule, the synthesized FeS2@BSA-Ce6 nanodots with the size of ~7 nm could be used for in vivo optical/ MR / PA tri-modal imaging, which achieved efficient accumulation within tumor sites after i.v. injection. Utilizing the good physical / chemical properties of FeS2@BSA-Ce6 nanodots, in vivo PTT & PDT combined therapies was then conducted, achieving synergistic tumor destruction without causing apparent in vivo toxicity.

2. Materials and methods 2.1 Materials Bovine serum albumin (BSA) was purchased from J&K Scientific Ltd. FeCl2, HfCl4, ErCl3, and Na2S were purchased from Shanghai Macklin Biochemical Co., Ltd. Chlorin e6 (Ce6) was purchased from Frontier Scientific Inc. 2.2 Synthesis of ultra-small FeS2 nanodots The

protein-modified

iron

sulfide

(FeS2)

nanodots

were

synthesized

by

bio-mineralization method according to the previous studies.36 35 mg BSA was


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dissolved in 9 mL H2O, into which FeCl2 (20 mM, 1mL) was slowly added under stirring. The pH was then adjusted to 6-7 by adding NaOH (1 M). Afterwards, 1mL Na2S solution (80 mM) was added to make the solution gradually turn dark green from pale yellow. After reaction at 25 oC for 2 h, the solution was collected for continuous dialysis for about 6 h until the final pH value was 6-7. Finally, the product was concentrated using an ultrafiltration device (MWCO, 100KDa) to obtain the FeS2@BSA solution ([Fe] = 10 mM). For the synthesis of FeS2: Hf@BSA and FeS2: Er@BSA ultra-small nanodots, 0.5 mL FeCl2 solution (20 mM) as well as 0.5 mL ErCl3 solution (20 mM) or HfCl4 solution (20 mM), respectively, was used to replace FeCl2 solution (20 mM) in the initial step. The following steps were identical to the synthesis of FeS2@BSA nanodots. 2.3 Ce6 conjugated on FeS2-BSA nanodots For Ce6 conjugation, 2 mg Ce6, along with 1 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma-Aldrich) and 1.5 mg sulfo-N-Hydroxysuccinimide (Sulfo-NHS, Sigma-Aldrich), was dissolved respectively in 100 µL DMSO and then mixed for activation for 1 h. 50 µL of activated Ce6 was added to 5 mL FeS2@BSA solution for stirring overnight. The supernatant was collected again after 14800 rpm centrifugation for 5 min. 2.4 Materials characterization The chemical composition was revealed by a PANalytical Empyrean X-ray Diffractometer. (High-resolution) TEM images as well as EDX of the nanomaterials were obtained using a Tecnai G2 F20 transmission electron microscope. The sizes and



the zeta potentials of nanomaterials were determined via a Zetasizer Nano Z (Malvern). The UV-vis-NIR spectrum was determined by a PerkinElmer Lambda 750 UV-vis-NIR spectrophotometer. The fluorescence of Ce6 was obtained by a TCSPC Fluorescence Spectrometer. The metal ions were determined by inductively coupled plasma mass spectrometry (ICP-MS) while the concentration of BSA was determined by a TGA1 Thermogravimetric Analysis (METTLE TOLEDO). The secondary structure of BSA in the nanomaterials was determined by a JASCO J-1500 circular dichroism. The singlet oxygen detection was followed the previous protocol using SOSG (Molecular Probes, USA).23 2.5 Cell culture experiments The standard 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was conducted to determine the murine breast cancer (4T1) cell viabilities. For in vitro PDT, 4T1 cells seeded in 96-well plates were incubated with FeS2@BSA or FeS2@BSA-Ce6 at various concentrations. PDT was conducted by exposing cells to the 660 nm xenon lamp (5 mW/cm2) after incubation for 12 h, whereas PTT was conducted by the 808 nm laser irradiation (0.8 W/cm2). The cells after various treatments were further stained by Calcein AM/propidium iodide (PI, Sigma-Aldrich). Cellular Ce6 fluorescence for cells incubated FeS2@BSA-Ce6 with or without laser irradiation (808 nm, 0.8 W/cm2, 20 min) was measured by a FACS Calibur Flow Cytometry (BD). For the detection of intracellular ROS, the cells were incubated with 2, 7-dichloro-dihydro-fluorescien diacetate (DCFH-DA, Sigma) for confocal fluorescence imaging.


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2.6 Tumor model All animal studies were conducted under a protocol approved by Soochow University Laboratory Animal Center. The 4T1 tumor model was generated by injecting cells (1 × 106) injected into the back of each nude mouse. 2.7 In vivo multimodal imaging For in vivo fluorescence imaging, 200 µL of FeS2@BSA-Ce6 with 3.5 mg/kg Ce6 and 12 mg/kg Fe2S was intravenously injected into each mouse. In vivo fluorescence imaging was conducted using an IVIS system at different time points. For in vivo PA imaging, mice were anesthetized and injected with FeS2@BSA-Ce6 at a dose of 3.5 mg/kg (in terms of Ce6). The tumor was imaged with a Vero LAIR animal imaging system at different time points. The magnetic properties of the nanodots were analyzed by a 1.0 T MR scanner. In vivo T2-weighted images were acquired using the previous parameters.30 2.8 In vivo cancer therapy Mice with the tumor volume reached ~70 mm3 were randomly divided into five groups (5 mice per group): (i) Control; (ii) FeS2@BSA-Ce6 only; (iii) FeS2@BSA-Ce6 + PDT; (iv) FeS2@BSA-Ce6 + PTT; and (v) FeS2@BSA-Ce6 + PTT + PDT. FeS2@BSA-Ce6 nanodots (at a dosage of 3.5 mg/kg Ce6 and 12 mg/kg FeS2) were intravenously injected into mice bearing 4T1 tumor. Phototherapy treatments were conducted 8 h later, with PTT by the 808 nm laser (0.8 W/cm2, 15 min), and (or) PDT by the 660 nm xenon lamp (5 mW/cm2, 30 min). Two days after different treatments, the tumors in each group were prepared for hematoxylin and eosin (H&E) ACS Paragon Plus Environment


staining. 2.9 Tumor slice staining The hypoxia staining of tumor slices together was conducted by the Hypoxyprobe-1 plus kit (Hypoxyprobe Inc) with blood vessel staining following the vendor’s protocol as described in previous studies.43 Prussian blue staining was carried out following the standard protocol to visualize iron contents within tumor slices, at 8 h after i. v. injection of FeS2@BSA-Ce6 (200 µL, 12 mg/kg in terms of FeS2).44

3. Results and discussion 3.1 Materials synthesis and characterization The protein-modified ultra-small iron sulfide (FeS2) nanodots were synthesized via the process of biomineralization at room temperature (Figure 1a). Briefly, bovine serum albumin (BSA) was chosen as a template which could adsorb Fe2+ ions. Afterwards, Na2S was added to trigger the nucleation and growth of FeS2 in albumin nanocages. The primary role of BSA was to provide binding sites, guide the nucleation and growth of FeS2@BSA nanodots, and then further provide various residues to conjugate with Ce6. Interestingly, the secondary structure of BSA was not greatly changed during this biomineralization processes (Supporting Figure S1). After removing excessive ions and unreactive reagents by dialysis and ultrafiltration, the synthesized nanoparticles were measured to be ~ 7 nm (Figure 1b, Supporting Figure S2). The FeS2@BSA nanodots were then characterized by X-ray diffraction (XRD) to confirm the chemical composition (Figure 1c). It was shown that all the


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diffraction peaks of XRD were in accordance with the FeS2 standard peaks (JCPDS No. 089-3057), demonstrating that the synthesized ultra-small nanodots were FeS2 nanomaterials. By this similar biomineralization method, some rare earth metal ions such as Hf and Er could be doped into FeS2 and form FeS2: Hf@BSA and FeS2: Er@BSA ultra-small nanodots (Supporting Figure S3), which may also be useful for bio-medical imaging and cancer therapy. We have done a series of experiments to change the ratio of BSA to iron (2: 1, 5: 1, and 10: 1) and found none size and morphology change of the FeS2 nanodots (Supporting Figure S4). In order to easily remove excess BSA for the next Ce6 conjugation step, we chose the low ratio of BSA: FeCl2 (2: 1) for the following experiments. The hydrodynamic size of the nanodots in aqueous solution was ~20 nm (Figure 1d), which was slightly larger than that measured by TEM image, probably due to the protein coating on the surface of FeS2 nanodots giving them good water solubility and biocompatibility. There was no need to further modify the as-prepared FeS2@BSA nanodots, since they could already have an excellent stability in various physiological solutions (Figure 1e). According to the thermogravimetric (TG) and inductively coupled plasma mass spectrometry (ICP-MS) analysis (Supporting Figure S5), the weight ratio of the BSA to FeS2 in the obtained FeS2@BSA nanodots was 2 : 3. As a result of the good NIR optical absorption of FeS2, those FeS2@BSA nanodots could act as an effective photothermal agent under NIR laser irradiation (Figure 2a). In order to further verify its thermal performance, FeS2@BSA nanodots



were irradiated by 808 nm laser for 5 min. It was found that the ultra-small FeS2@BSA nanodots showed great concentration-dependent heating effect upon NIR laser irradiation (Figure 2b). The photothermal stability, which was also an important index of photothermal reagents, was measured in three cycles, with no noticeable change in the photothermal stability of FeS2@BSA observed (Supporting Figure S6). In a word, the synthesized FeS2@BSA ultra-small nanodots have good NIR absorption and photothermal stability, which are desired for their applications as a new photothermal reagent. We next investigated the magnetic properties of FeS2@BSA nanodots. T2-weighted MR images of FeS2@BSA nanodots showed a concentration-dependent darkening effect (Figure 2c, Supporting Figure S7). Based on the Fe concentration, the r2 relaxivity of FeS2@BSA nanodots was measured to be 85.36 mM S−1, which was slightly lower than that of iron oxide nanoparticles (IONPs, 105.93 mM S−1), but still high enough for T2 weighted MR imaging. Since the surface of the protein modified FeS2 nanodots were rich in functional groups such as amino groups, Ce6 could be further coupled to BSA for fluorescent imaging and PDT through the amide coupling method. Different amounts of Ce6 molecules were covalently attached to the surface of FeS2@BSA. After removing excess Ce6 molecules by centrifugation, the supernatant was collected to obtain FeS2@BSA-Ce6 nanocomplex. In the UV-vis-NIR absorption spectra before and after coupling PS molecules (Figure 2d), the Ce6 absorption peak was enhanced with the increase of Ce6 content, indicating that Ce6 was successfully conjugated to


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FeS2@BSA nanodots (Figure 2e). The final molar ratio of FeS2: Ce6 used for the following experiment was 10 : 1 (Actual loading capacity). After conjugation, we did not find any quenching effect for the final FeS2@BSA-Ce6 nanodots (Supporting Figure S8), whose MR contrast was also not affected (Figure 2c). The singlet oxygen generation ability of FeS2@BSA-Ce6 could be detected using singlet oxygen sensor green (SOSG) probe. Under the 660 nm light irradiation, the fluorescence of SOSG incubated with FeS2@BSA-Ce6 increased gradually, suggesting the light-triggered production of singlet oxygen (Figure 2f). Compared with Ce6, FeS2@BSA-Ce6 nanodots at the same Ce6 concentration showed the similar efficiency of light-triggered singlet oxygen production, indicating that the synthesized FeS2@BSA-Ce6 nanodots could be used as an effective PDT agent.

3.2. In vitro experiments We then performed the PDT treatment of 4T1 murine breast cancer cells. After 24 h incubation with different concentrations of FeS2@BSA or FeS2@BSA-Ce6 without light irradiation (Figure 3a), all of them didn’t show any toxicity to the cells even at a high concentration of FeS2@BSA ([Fe] = 0.04 mM), suggesting the good biocompatibility of those nanodots. After 24 h incubation with FeS2@BSA or FeS2@BSA-Ce6 in the presence of 660 nm light exposure, the cells incubated with FeS2@BSA-Ce6 nanodots could be effectively killed by a concentration-dependent manner after PDT, while the cell viability of the control group with FeS2@BSA incubation didn’t show significant change after light exposure.



To trigger in vitro PTT, FeS2@BSA or FeS2@BSA-Ce6 were incubated with cells and exposed to an 808 nm laser for 20 min. The cell viability assay clearly illustrated the concentration-dependent photothermal cancer cell killing effect (Figure 3b). According to the recent studies, mild hyperthermia could increase cell permeability, and further promote the effect of nanoparticle-mediated cancer therapy.19 To test whether the heat generated by FeS2 nanodots could be used to promote the cell uptake and thus improve the efficacy of PDT, cells were cultured with FeS2@BSA or FeS2@BSA-Ce6 nanoparticles under the condition of the laser irradiation or not (808 nm, 0.8 W/cm2, 20 min), which could generate moderate heating and raise the temperature of the medium to around 43 oC (Figure 3g). At the cellular level, the therapeutic effect of PDT and PTT combined therapy was further investigated. Compared with the control group, the combined treatment showed the best cell killing ability (Figure 3c&d). We also used DCFH-DA probe to detect the ROS generation during PDT and PDT & PTT. It could be found the ROS generation was much stronger under photothermal effect, probably for the reason that the mild hyperthermia could increase cell permeability and the concentration of Ce6 accumulated inside the cells (Figure 3e). To measure the cell uptake of Ce6 under different conditions quantitatively, a FACS Calibur Flow Cytometry (BD) was employed. The fluorescence of laser-treated FeS2@BSA-Ce6-incubated cells appeared to be much stronger (Figure 3f), whereas FeS2@BSA-Ce6-incubated cells without irradiation showed rather weak fluorescence. Therefore, the mild PTT could indeed increase the efficiency of PDT treatment at the cellular level, by increasing the


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cellular uptake level of FeS2@BSA-Ce6 nanodots.

3.3. In vivo multimodal imaging Utilizing the strong NIR absorbance and high r2 relaxivity of the synthesized ultra-small FeS2 nanodots, as well as the Ce6 fluorescent molecule conjugated on the FeS2@BSA-Ce6, next we would like to investigate the in vivo behavior of FeS2-Ce6 nanodots using different imaging modalities. Firstly, the fluorescence of FeS2@BSA-Ce6 could be used to study the in vivo behavior of nanodots. The fluorescence signals of the tumor region increased and reached the peak at 8 h after i.v. injection (Figure 4a), suggesting the effective tumor uptake of those nanodots by the enhanced permeability and retention (EPR) effect (Figure 4b). As time goes on, we found that the tumor fluorescence signals showed a decrease, probably due to the partial clearance of those ultra-small FeS2@BSA-Ce6 nanodots. Semi-quantitative biodistribution further evidenced the efficient tumor accumulation of those ultra-small FeS2@BSA-Ce6 nanodots (Supporting Figure S9), in good agreement with the images of Prussian blue staining (Supporting Figure S10). Strong fluorescence signal was observed from the kidney after 24 h p. i., indicating the possible renal clearance of those ultra-small nanodots. With strong NIR absorbance, FeS2@BSA-Ce6 could also be tracked by PA imaging, which was conducted for mice after i.v. injection of FeS2@BSA-Ce6 (200 µL, 3.5 mg/kg) (Figure 4c). The PA signals of the tumor region gradually increased with time, clearly demonstrating the EPR enrichment of FeS2@BSA-Ce6 nanodots in



the tumor, consistent with the above fluorescence imaging (Figure 4d). Inspired by the T2 contrast performance of FeS2, in vivo MR imaging was then conducted for 4T1 tumor-bearing mice before and post intravenous injection of FeS2@BSA-Ce6 nanodots (200 µL, 5 mg/kg) (Figure 4e&f). The enhanced MR contrast at the tumor site (yellow dashed circle) was observed at 8 h p.i, further demonstrating the efficient tumor accumulation of those nanodots. From the above results, our FeS2@BSA-Ce6 nanodots have realized optical / PA / MR triple-modal imaging. Taking advantages of the unique advantages of each imaging modality, the combined imaging would provide valuable information for cancer therapy.25, 39, 40

3.4 In vivo combined tumor therapy Next, we investigated the in vivo NIR-induced phototherapy performance of FeS2@BSA-Ce6 nanodots. The tumor temperature under the laser irradiation could be monitored by an infrared thermal camera (Figure 5a). Under the 808 nm laser irradiation, FeS2@BSA-Ce6 accumulated in the tumor converted the absorbed light energy into heat, and the temperature of the tumor would be increased from 30.5 oC to 46 oC (Figure 5b). In contrast, the tumor of mice injected with PBS showed much less significant temperature increase. According to the previous work, the mild photothermal effect may enhance blood flow into the tumor and then improve overall tumor oxygenation status.30, 41, 42 A hypoxyprobe immunofluorescence assay was then conducted on tumor slices of the mice, further demonstrating the efficacy of PTT to improve the hypoxia inside the tumor (Figure 5c).


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With the support of imaging data, we further studied the in vivo therapeutic effect of FeS2-Ce6 nanodots. Mice with 4T1 tumors were randomly divided into 5 groups (5 mice per each group): (i) The control; (ii) FeS2@BSA-Ce6 nanodots injection; (iii) FeS2@BSA-Ce6 nanodots injection + PDT; (iv) FeS2@BSA-Ce6 nanodots injection + PTT; (v) FeS2@BSA-Ce6 nanodots injection + PTT + PDT. PTT or PDT was conducted at 8 h post injection of the nanodots, using either the 808 nm laser (0.8 W/cm2 for 15 min) or 660 nm xenon lamp (5 mW/cm2 for 30 min). Remarkably, the tumors on FeS2@BSA-Ce6 treated mice completely disappeared after the combined PTT + PDT treatment (Figure 5d&e), in marked contrast to the other groups in which PDT alone or PTT alone could slightly inhibit the tumor growth (Figure 5e, Supporting Figure S11). Tumor slices were stained by hematoxylin and eosin (H&E) to further assess the therapy effect with different treatments (Figure 5f). While the tumors from FeS2-Ce6 nanodots treated mice were only partially destroyed after PTT or PDT alone, the combination PTT + PDT therapy with FeS2-Ce6 nanodots could lead to severe destruction of tumor cells. During the whole treatment process, no apparent toxicity of FeS2@BSA-Ce6 nanodots was found after i.v. injection. No sudden death or unexpected body weight loss was observed after the combined PTT + PDT treatment with FeS2@BSA-Ce6. Finally, major organs were collected for H&E staining (Supporting Figure S12). We did not find any obvious organ damage within 30 days after i.v. injection of FeS2@BSA-Ce6 nanodots, indicating the great biocompatibility of such albumin modified ultra-small FeS2 nanodots.



4. Conclusions In summary, a protein-assisted synthesis method was developed to prepare ultra-small FeS2 nanodots for imaging-guided combined cancer therapy. Taking advantage of the strong NIR absorbance and high r2 relaxivity of the synthesized ultra-small FeS2 nanodots, as well as the inherent Ce6 fluorescence from FeS2@BSA-Ce6 nanodots, in vivo optical / PA / MRI tri-modal imaging was carried out, revealing efficient tumor accumulating of those nanodots after i.v. injection. We further employed FeS2@BSA-Ce6 nanodots for combined PTT and PDT, which resulted in a remarkably synergistic tumor destruction effect, probably due to the moderate photothermal effect to promote the efficacy of PDT via relieving tumor hypoxia, as well as enhancing cellular uptake of Ce6 conjugated nanodots. Our study thus provides a new type of ultra-small nanodots with multiple functionalities integrated within a single nanoscale system, useful for multimodal biomedical imaging-guided combined cancer therapy.

ASSOCIATED CONTENT The authors declare no competing financial interest.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website.


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Materials characterization, ex vivo data, Prussian blue staining, photos of tumor therapy, and body weight of the various treatments (PDF).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (L. Cheng)

Acknowledgement

This work was partially supported by the National Natural Science Foundation of China (51572180, 51525203, 51302180), the National Research Programs from Ministry of Science and Technology (MOST) of China (2016YFA0201200), a Jiangsu Natural Science Fund for Young Scholars (BK20170063, BK20130005), and the Post-doctoral science foundation of China (2013M531400, 2014T70542).

Reference 1. Galluzzi, L.; Kepp, O.; Vander Heiden, M. G.; Kroemer, G. Metabolic Targets for Cancer Therapy. Nat. Rev. Dru Discovery 2013, 12, 829-846. 2. Murtaza, M.; Dawson, S. J.; Tsui, D. W.; Gale, D.; Forshew, T.; Piskorz, A. M.; Parkinson, C.; Chin, S. F.; Kingsbury, Z.; Wong, A. S. Non-invasive Analysis of Acquired Resistance to Cancer Therapy by Sequencing of Plasma DNA. Nature 2013, 497, 108-112. 3. Cheng, C. J.; Bahal, R.; Babar, I. A.; Pincus, Z.; Barrera, F.; Liu, C.; Svoronos, A.; Braddock, D. T.; Glazer, P. M.; Engelman, D. M. MicroRNA Silencing for Cancer Therapy Targeted to the Tumour Microenvironment. Nature 2015, 518, 107-110. 4. Altman, B. J.; Stine, Z. E.; Dang, C. V. From Krebs to Clinic: Glutamine Metabolism to Cancer Therapy. Nat. Rev. Cancer 2016, 16, 619-634. 5. Topalian, S. L.; Drake, C. G.; Pardoll, D. M. Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy. Cancer cell 2015, 27, 450-461.



6. Miest, T. S.; Cattaneo, R. New Viruses for Cancer Therapy: Meeting Clinical Needs. Nat. Rev. Microbiol. 2014, 12, 23-34. 7. Schumacher, T. N.; Schreiber, R. D. Neoantigens in Cancer Immunotherapy. Science 2015, 348, 69-74. 8. Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869-10939. 9. Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as An Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751-760. 10. Skrabalak, S. E.; Chen, J.; Sun, Y.; Lu, X.; Au, L.; Cobley, L. M.; Xia, Y. Gold Nanocages: Synthesis, Properties, and Applications. Acc. Chem. Res. 2008, 41, 1587-1595. 11. Xie, J.; Lee, S.; Chen, X. Nanoparticle-Based Theranostic Agents. Adv. Drug Delivery Rev. 2010, 62, 1064-1079. 12. Wang, Y.; Black, K. C.; Luehmann, H.; Li, W.; Zhang, Y.; Cai, X.; Wan, D.; Liu, S.-Y.; Li, M.; Kim, P. A Comparison Study of Gold Nanohexapods, Nanorods, and Nanocages for Photothermal Cancer Treatment. ACS Nano 2013, 7, 2068-2077. 13. Huang, P.; Lin, J.; Wang, X.; Wang, Z.; Zhang, C.; He, M.; Wang, K.; Chen, F.; Li, Z.; Shen, G. Light Triggered Theranostics based on Photosensitizer Conjugated Carbon Dots for Simultaneous Enhanced Fluorescence Imaging and Photodynamic Therapy. Adv. Mater. 2012, 24, 5104-5110. 14. Xu, P.; Van Kirk, E. A.; Zhan, Y.; Murdoch, W. J.; Radosz, M.; Shen, Y. Targeted Charge Reversal Nanoparticles for Nuclear Drug Delivery. Angew. Chem. Inter. Ed. 2007, 46, 4999-5002. 15. Yue, X.; Zhang, Q.; Dai, Z. Near Infrared Light Activatable Polymeric Nanoformulations for Combined Therapy and Imaging of Cancer. Adv. Drug Delivery Rev.2017, 115, 155-170. 16. Chen, Q.; Xu, L.; Liang, C.; Wang, C.; Peng, R.; Liu, Z. Photothermal Therapy with Immune-Adjuvant Nanoparticles together with Checkpoint Blockade for Effective Cancer Immunotherapy. Nat. Commun. 2016, 7, 13193. 17. Wang, C.; Sun, W.; Ye, Y.; Hu, Q.; Bomba, H. N.; Gu, Z. In Situ Activation of Platelets with Checkpoint Inhibitors for Post-Surgical Cancer Immunotherapy. Nat. Biomed. Eng. 2017, 1, 0011. 18. Lin, J.; Wang, S.; Huang, P.; Wang, Z.; Chen, S.; Niu, G.; Li, W.; He, J.; Cui, D.; Lu, G. Photosensitizer-Loaded Gold Vesicles with Strong Plasmonic Coupling Effect for Imaging Guided Photothermal/ Photodynamic Therapy. ACS Nano 2013, 7, 5320-5329. 19. Gong, H.; Dong, Z.; Liu, Y.; Yin, S.; Cheng, L.; Xi, W.; Xiang, J.; Liu, K.; Li, Y.; Liu, Z. Engineering of Multifunctional Nano Micelles for Combined Photothermal and Photodynamic Therapy under the Guidance of Multimodal Imaging. Adv. Funct. Mater. 2014, 24, 6492-6502. 20. Tian, B.; Wang, C.; Zhang, S.; Feng, L.; Liu, Z. Photothermally Enhanced Photodynamic Therapy Delivered by Nano-Graphene Oxide. ACS Nano 2011, 5, 7000-7009.


Page 18 of 27

Page 19 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21. Lucky, S. S.; Soo, K. C.; Zhang, Y. Nanoparticles in Photodynamic Therapy. Chem. Rev. 2015, 115, 1990-2042. 22. Ge, J.; Lan, M.; Zhou, B.; Liu, W.; Guo, L.; Wang, H.; Jia, Q.; Niu, G.; Huang, X.; Zhou, H. A Graphene Quantum Dot Photodynamic Therapy Agent with High Singlet Oxygen Generation. Nat. Commun. 2014, 5, 4596. 23. Cheng, L.; Kamkaew, A.; Sun, H.; Jiang, D.; Valdovinos, H. F.; Gong, H.; England, C. G.; Goel, S.; Barnhart, T. E.; Cai, W. Dual Modality Positron Emission Tomography / Optical Image-Guided Photodynamic Cancer Therapy with Chlorin e6-Containing Nanomicelles. ACS Nano 2016, 10, 7721-7730. 24. Lovell, J. F.; Liu, T. W.; Chen, J.; Zheng, G. Activatable Photosensitizers for Imaging and Therapy. Chem. Rev. 2010, 110, 2839-2857. 25. Lee, D. E.; Koo, H.; Sun, I. C.; Ryu, J. H.; Kim, K.; Kwon, I. C. Multifunctional Nanoparticles for Multimodal Imaging and Theragnosis. Chem. Soc. Rev. 2012, 41, 2656-2672. 26. Cheng, L.; Yang, K.; Li, Y.; Chen, J.; Wang, C.; Shao, M.; Lee, S. T.; Liu, Z. Facile Preparation of Multifunctional Upconversion Nanoprobes for Multimodal Imaging and Dual‐Targeted Photothermal Therapy. Angew. Chem. Int. Ed. 2011, 123, 7523-7528. 27. Gai, S.; Li, C.; Yang, P.; Lin, J. Recent Progress in Rare Earth Micro/Nanocrystals: Soft Chemical Synthesis, Luminescent Properties, and Biomedical Applications. Chem. Rev. 2013, 114, 2343-2389. 28. Meng, Z.; Wei, F.; Ma, W.; Yu, N.; Wei, P.; Wang, Z.; Tang, Y.; Chen, Z.; Wang, H.; Zhu, M. Design and Synthesis of “All in One” Multifunctional FeS2 Nanoparticles for Magnetic Resonance and Near Infrared Imaging Guided Photothermal Therapy of Tumors. Adv. Funct. Mater. 2016, 26, 8231-8242. 29. Yang, K.; Yang, G.; Chen, L.; Cheng, L.; Wang, L.; Ge, C.; Liu, Z. FeS Nanoplates as A Multifunctional Nano Theranostic for Magnetic Resonance Imaging Guided Photothermal Therapy. Biomaterials 2015, 38, 1-9. 30. Cheng, L.; Shen, S.; Shi, S.; Yi, Y.; Wang, X.; Song, G.; Yang, K.; Liu, G.; Barnhart, T. E.; Cai, W. FeSe2 Decorated Bi2Se3 Nanosheets Fabricated via Cation Exchange for Chelator Free 64Cu Labeling and Multimodal Image Guided Photothermal Radiation Therapy. Adv. Funct. Mater. 2016, 26, 2185-2197. 31. Choi, H. S.; Liu, W.; Liu, F.; Nasr, K.; Misra, P.; Bawendi, M. G.; Frangioni, J. V. Design Considerations for Tumour-Targeted Nanoparticles. Nat. Nanotechnol. 2010, 5, 42-47. 32. Zhou, C.; Long, M.; Qin, Y.; Sun, X.; Zheng, J. Luminescent Gold Nanoparticles with Efficient Renal Clearance. Angew. Chem. Inter. Ed. 2011, 50, 3168-3172. 33. Liu, F.; He, X.; Chen, H.; Zhang, J.; Zhang, H.; Wang, Z. Gram Scale Synthesis of Coordination Polymer Nanodots with Renal Clearance Properties for Cancer Theranostic Applications. Nat. Commun. 2015, 6, 8003. 34. Cölfen, H. Biomineralization: A Crystal-Clear View. Nat. Mater. 2010, 9, 960-961. 35. Mao, L. B.; Gao, H. L.; Yao, H. B.; Liu, L.; Cölfen, H.; Liu, G.; Chen, S. M.; Li, S. K.; Yan, Y. X.; Liu, Y. Y. Synthetic Nacre by Predesigned Matrix-Directed



Mineralization. Science 2016, 354, 107-110. 36. Yang, T.; Wang, Y.; Ke, H.; Wang, Q.; Lv, X.; Wu, H.; Tang, Y.; Yang, X.; Chen, C.; Zhao, Y. Protein Nanoreactor Assisted Synthesis of Semiconductor Nanocrystals for Efficient Cancer Theranostics. Adv. Mater. 2016, 28, 5923-5930. 37. Yang, W.; Guo, W.; Le, W.; Lv, G.; Zhang, F.; Shi, L.; Wang, X.; Wang, J.; Wang, S.; Chang, J.; Zhang, B. Albumin Bioinspired Gd:CuS Nanotheranostic Agent for In Vivo Photoacoustic/Magnetic Resonance Imaging-Guided Tumor-Targeted Photothermal Therapy. ACS Nano 2016, 10, 10245-10257. 38. Zhu, A.; Miao, K.; Deng, Y.; Ke, H.; He, H.; Yang, T.; Guo, M.; Li, Y.; Guo, Z.; Wang, Y. Dually pH/ Reduction-Responsive Vesicles for Ultrahigh Contrast Fluorescence Imaging and Thermo Chemotherapy Synergized Tumor Ablation. ACS Nano 2015, 9, 7874-7885. 39. Kim, J.; Piao, Y.; Hyeon, T. Multifunctional Nanostructured Materials for Multimodal Imaging, and Simultaneous Iimaging and Therapy. Chem. Soc. Rev. 2009, 38, 372-390. 40. Sun, X.; Cai, W.; Chen, X. Positron Emission Ttomography Imaging Using Radiolabeled Inorganic Nanomaterials. Acc. Chem. Res. 2015, 48, 286. 41. Cheng, L.; Yuan, C.; Shen, S.; Yi, X.; Gong, H.; Yang, K.; Liu, Z. Bottom up Synthesis of Metal Ion Doped WS2 Nanoflakes for Cancer Theranostics. ACS Nano 2015, 9, 11090-11101. 42. Song, G.; Liang, C.; Gong, H.; Li, M.; Zheng, X.; Cheng, L.; Yang, K.; Jiang, X.; Liu, Z. Core shell MnSe@ Bi2Se3 Fabricated via A Cation Exchange Method as Novel Nanotheranostics for Multimodal Imaging and Synergistic Thermoradiotherapy. Adv. Mater. 2015, 27, 6110-6117.


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Figure 1. Synthesis and characterization of FeS2 ultra-small nanodots. (a) Synthetic route and applications of FeS2 nanodots. (b) TEM image of FeS2 nanodots. Inset: High-resolution TEM image of FeS2 nanodots. (c) XRD of the synthesized FeS2 ultra-small nanodots. (d) Hydrodynamic size of FeS2@BSA nanodots in aqueous solution. (e) A photo of FeS2@BSA nanodots in various solutions: water, phosphate buffered saline (PBS), 1640 cell medium, and fetal bovine serum (FBS).



Figure 2. Optical and magnetic properties of the FeS2@BSA ultra-small nanodots. (a) UV-vis-NIR absorbance spectra of FeS2@BSA nanodots. (b) Heating curves of FeS2@BSA solutions at di‐erent concentrations under irradiation by an 808 nm laser (0.8 W/cm2, 5 min). (c) The relative relaxation rate r2 of FeS2@BSA nanodots, FeS2@BSA-Ce6 nanocomplex, and iron oxide nanoparticles (IONPs), respectively. (d&e) UV-vis-NIR spectra of FeS2@BSA at different Ce6 feeding concentrations (Fe: Ce6 = 8 : 1, 4 : 1, 2 : 1, 1 : 1) (d) and loading efficiency respectively (e). (f) The generation of singlet oxygen determined by the increased SOSG fluorescence from H2O, FeS2@BSA, Ce6, and FeS2@BSA-Ce6 nanocomplex, n=3.


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Figure 3. In vitro phototherapy. (a-c) Cell viability data obtained from the MTT assay of 4T1 cells after various treatments indicated. Relative cell viabilities in all samples were normalized to the control saline-added samples without laser irradiation (100% viability). Error bars were based on SD of at least four parallel samples. (a) Cells treated with FeS2@BSA-Ce6 and Ce6 without laser irradiation for 24 h. (b) Cells treated with FeS2@BSA-Ce6 without and with irradiation by the 808 nm laser (0.8 W/cm2, 20 min). (c) Cells treated with FeS2@BSA-Ce6 with irradiation by the 660 nm xeon lamp (5 mW/cm2, 30 min), the 808 nm laser (0.8 W/cm2, 20 min) or both. (d) Confocal images of calcein AM (green, live cells) and propidium iodide (red, dead cells) co-stained cells after incubation with FeS2@BSA-Ce6 nanoparticles for different phototherapy. (e) Confocal images of intracellular ROS generation in 4T1 cells using DCFH-DA assay. Green channel emission was collected at 480-520 nm upon excitation at 488 nm. (f) Flow cytometry measurement of cellular Ce6 fluorescence for cells incubated FeS2@BSA-Ce6 with or without laser irradiation (808 nm, 0.8 W/cm2, 20 min). P values: ***p < 0.001, **p < 0.01, or *p < 0.05. (g) A scheme showing photothermal enhanced cellular uptake of FeS2@BSA-Ce6 nanoparticles.



Figure 4. In vivo multimodal imaging. (a&b) In vivo fluorescence images (a) and fluorescence intensities (b) of 4T1 tumor-bearing nude mice taken at different time points post i.v. injection of FeS2@BSA-Ce6 nanodots (3.5 mg/kg Ce6 and 12 mg/kg FeS2). (c&d) Photoacoustic images (c) and photoacoustic signal (d) of tumors in mice taken at different time points post i.v. injection of FeS2@BSA-Ce6 nanodots (3.5 mg/kg Ce6 and 12 mg/kg FeS2). (e&f) MR images (e) and quantified MR signals (f) of 4T1 tumor-bearing nude mice before and 8 h after i.v. injection of FeS2@BSA-Ce6 nanodots (3.5 mg/kg Ce6 and 12 mg/kg FeS2). P values: ***p < 0.001, **p < 0.01, or *p < 0.05.


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Figure 5. In vivo combined therapy. (a) IR thermal images of tumors in mice i.v. injected with FeS2@BSA-Ce6 under 808 nm laser irradiation (0.8 W/cm2, 15 min). (b) Surface temperature changes of tumors monitored by the IR thermal camera during laser irradiation. (c) Representative immunofluorescence images of tumor slices after hypoxia staining. The cell nucleus, hypoxia areas, and blood vessels were stained with DAPI (blue), anti-pimonidazole antibody (green) and anti-CD31 antibody (red), respectively. (d) Relative tumor volume curves of different groups of mice after the various treatments indicated. Five mice were used in each group. Error bars are based



on standard errors of the mean. (e) Photograph of tumors collected from different groups of mice 14 days after the treatment was initiated. (f) H&E stained tumor slices from different groups of mice treated with PBS, FeS2@BSA-Ce6, FeS2@BSA-Ce6 + PDT, FeS2@BSA-Ce6 + PTT, and FeS2@BSA-Ce6 + PTT + PDT, respectively. The tumors were harvested 2 days after treatments were conducted.


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Albumin Assisted Synthesis of Ultra-small FeS2 ... - ACS Publications

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