One-Pot Synthesis of a Bismuth Selenide Hexagon Nanodish

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One-pot synthesis bismuth selenide hexagon nanodish complex for multi-modal imaging-guided combined antitumor phototherapy Yilin Song, Jinping Wang, Li Liu, Qi Sun, Qing You, Yu Cheng, Yidan Wang, Siyu Wang, Fengping Tan, and Nan Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00106 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

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Molecular Pharmaceutics

One-pot synthesis bismuth selenide hexagon nanodish complex for multi-modal imaging-guided combined antitumor phototherapy Yilin Song, Jinping Wang, Li Liu, Qi Sun, Qing You, Yu Cheng, Yidan Wang, Siyu Wang, Fengping Tan, Nan Li* Tianjin Key Laboratory of Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, 300072, Tianjin, PR China.

*Corresponding author at: School of Pharmaceutical Science and Technology, Tianjin University, 300072, Tianjin, PR China. Tel.:+86-022-27404986 E-mail address: [email protected]

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Abstract For integrating therapy and diagnose into a single nanoparticle for higher anti-tumor efficiency and lower toxicity, our group designed a smart theranostic nanoplatform based on a hyaluronic acid-doped polypyrrole coated bismuth selenide loading with zinc phthalocyanine nanodish complex (Bi2Se3@HA-doped PPy/ZnPc) for multi-modal imaging guided combined phototherapy. Moreover, we expect that HA-doped PPy smart shell for surface functionalization will be also applied to a variety of 2D nanomaterials shared the similar structure of Bi2Se3 to broader their applications in biomedicine. Bi2Se3 hexagon nanodish was synthesized via a simple and safe solution-based method compared to common adopted ones. One-pot synthesis of naoncomplex was carried out by adding HA during polypyrrole coating on Bi2Se3 process, then further loaded with ZnPc. Besides the good ability for infrared thermal, photoacoustic, fluorescence and X-ray computed tomography imagings, nanodish complex own high photo-heat conversion efficiency for photothermal therapy and remarkable optical absorption coefficient for photodynamic therapy. With EPR effect of nanoparticles and CD44-targeted effect of HA, tumor-growth inhibition ratio of Bi2Se3@HA-doped PPy/ZnPc for PTT-PDT was as high as 96.4%, compared with the PTT (68.0%) or PDT (27.3%) alone, showing an excellent combined therapeutic effect. Moreover, no obvious toxicity in vivo was caused by the nanoparticles. Thus, such Bi2Se3@HA-doped PPy/ZnPc nanodish complex is hopeful for real-time monitoring and precise, high-efficient antitumor treatment.

Keywords: Theranostic, Bismuth selenide hexagon nanodish, One-pot synthesis, Surface functional, Triple-modal imaging-guided, Phototherapy.

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Abbreviations: Bi2Se3

Bismuth selenide

HA

Hyaluronic acid

PPy

Polypyrrole

ZnPc

Zinc phthalocyanine

Bi2Se3@HA-doped PPy BPH

Hyaluronic acid-doped polypyrrole coated bismuth selenide

Hyaluronic acid-doped polypyrrole coated bismuth selenide

Bi2Se3@HA-doped PPy/ZnPc

Hyaluronic acid-doped polypyrrole coated bismuth

selenide loading with zinc phthalocyanine BPHZn

Hyaluronic acid-doped polypyrrole coated bismuth selenide loading with

zinc phthalocyanine CT

X-ray computed tomography

PA

Photoacoustic

NIRF

Near-infrared fluorescence

NIR

Near-infrared

PTT

Photothermal therapy

PDT

Photodynamic therapy

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Introduction Today, multi-functional nanoparticles offering both diagnostic and therapeutic functions have draw huge attention. Particularly, nanoparticle-mediated spatially precise photothermal therapy (PTT) combined with real-time monitoring has draw recent

attention

because

of

targeting,

remote-control,

simplicity

likewise

noninvasiveness and safety performance.(1-5) What’s more, the imaging materials allows identifying tumors’ size as well as location, detecting the accumulation of PT agents and monitoring the effect of therapy.(6-8) But an expensive, sophisticated, and challenging synthesis is usually asked for integrating photothermal agents with other components, such as high-Z elements for X-ray-computed tomography, magnetic materials for magnetic resonance imaging, and upconversion luminescence nanoparticles or fluorescence molecule for fluorescence imaging.(9-13) Under certain conditions, to overcome the limitations of single modality, even multiple ingredient for different imaging modal are required. (4,5) Shortcomings of such assembling have been disclosed: different dose asked for therapy and imaging functions, compatibility of the composites and uncertain stability, high toxicity in vivo, and even diverse dissociation rate of different part during systemic circulation.(14-18) Therefore, it is important to build multi-functional nanoparticles possessing both imaging and therapeutic functions. Among a large amount of nanocarriers, such 2D layered materials as graphene have been payed much attention because of their distinct properties and promising applications in many fields including pharmaceutics.(19,20) As a typical 2D layered nanomaterial, bismuth selenide (Bi2Se3) has remarkable photoelectric and thermoelectric properties.(21-24) Recently, some papers reported that the Bi2Se3 nanoparticles is bio-compatible, metabolizable, and the low toxic in vivo.(19) And more important, the promising usage of Bi2Se3 nanoparticles for PTT and CT, PA imaging were also unveiled.(25,26) Among the different shape of Bi2Se3 nanoparticles, such as Bi2Se3 nanoplate, nanosheet, nanoscale spherical sponge et al.,(26-28) smaller and thinner Bi2Se3 nanoparticles is considered to improve the optical absorption

4


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properties and excretion from the body after systemic administration efficiently. However, in previous synthesis methods, toxic Na2SeO3 is usually required as the Se source. In vivo and vitro instability and high oxidation is present Bi2Se3 nanoagents’ another major challenge.(19) Such drawbacks have largely limited Bi2Se3 nanoagents’ practical usage. Nevertheless, the use of Bi2Se3 nanoagents as drug carriers is also limited by lacking proper surface functionalization and the difficulty of following drug loading. In this work, we prepared a thin hexagon nanodish-shaped Bi2Se3 by a simple solution-based method using Se powders instead of toxicity NaHSe as Se source for the first time. In addition, to overcome the disadvantages of instability and drug loading capability, we introduced hyaluronic acid (HA)-doped polypyrrole (PPy) coated bismuth selenide (Bi2Se3) hexagon nanodish (BPH) in this project. HA could target cancer cells through active and passive targeting pathway, which respectively due to specifically overexpressed lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) or cluster determinant 44 (CD44) which bound to cancer cells.(29,30) and the enhanced permeation and retention (EPR) effect. Through receptor-mediated endocytosis, BPH NPs will be uptaked by tumor cells, decomposed into shorter parts by hyaluronidases (mainly Hyal-1 and Hyal-2)(31,32) then released the loading inside the cell. In addition, it was reported that, PPy may have great possibility as an efficient energy quenchers, for its immense absorption coefficient (around 105-fold higher than traditional organic fluorochromes) with a broader peak(33). On the other hand, due to light scattering and biological tissues absorption, eradicating tumors completely by single PTT is hard. Thus, combined PTT and PDT is catched for optimized and enhanced and tumor killing efficacy.(34) In turn, PTT could boost blood streams and bring more oxygen into tumor site, to overcome hypoxia-associated PDT resistance. As a promising photosensitizer for PDT(35-37), ZnPc possess excellent optical absorption coefficient in the 600-800 nm photodynamic therapeutic window ( the coefficient of normal used porphyrin is much lower) (38-41). Due to hydrophobic and self-aggregation properties, ZnPc is largely limited its applications(42). In order to avert of ZnPc aggregated in aqueous solutions, 5



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we load ZnPc onto BPH. We hypothesize that, once ZnPc are near to the PPy, efficient energy transfer from ZnPc to PPy, fluorescence of ZnPc will be quenched (OFF). After loading onto the BPH, the fluorescence of ZnPc remains “off” during blood circulating. After internalizing into tumor cells via receptor-mediated pathway, hyaluronidases (mainly Hyal-1 and Hyal-2) decomposed the nanoparticles into parts and exposed the ZnPc inside the cell, thus resulting in fluorescence activation. Therefore, in contrast to free ZnPc with always “on” fluorescence and unfavorable pharmacokinetic behavior, BPHZn nanoparticles have utility in real-time monitor: high target-to-background ratio fluorescence imaging are important for lower systemic toxic cancer therapy. (43) Thus, we have designed and synthesised a multi-functional nanoagent for PT and PD combined therapy and triple-modal imaging using hyaluronic acid (HA)-doped polypyrrole (PPy) coated bismuth selenide (Bi2Se3)

hexagon

nanodish

loaded

with

zinc

phthalocyanine

(ZnPc)

(Bi2Se3@HA-doped PPy/ZnPc). We demonstrated that 1) Bi2Se3 nanodish here with a thickness down to few quantum layers was expected to have higher optical absorption abilities and excrete from the body efficiently after systemic administration. 2) The coating was synthesized in only one step and would successfully protect the Bi2Se3 from oxidation, keep stable and dissoluble in different physical solutions during a long time. 3) The fluorescence of ZnPc was quenched when loaded on the BPH nanoparticles and dequenched after entering into the cancer cells, therefore realizing the high target-to-background ratio real-time moniter fluorescence imaging. 4) In addition to the bimodal targeted ability, excellent CT, photoacoustic and fluorescence performance, the strong absorption in NIR region, remarkable stability of photothermal conversion and photodynamic ability, supported the application of the BPHZn nanoparticles for triple-imaging guided combined PPT and PDT treatment. CT imaging has some known advantages such as, facile three-dimensional visualization and higher resolution compared with other imaging modes. However CT has its own limitations, such as poor contrast in soft tissues and low sensitivity. Photoacoustic has become a crutial imaging mode offering high soft tissues spatial 6


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resolution and overcome shortages of CT. With the low toxicity and high biocompatibility, this functionalized Bi2Se3 based multi-functional nanoparticle is very favorable and promising for safe, effective, and efficient antitumor therapy in clinic. And we expect that our surface functionalzed strategy will be applied to a variety of 2D nanomaterials shares the similar stucture to promote their broad applications in biomedicine too.

2. Materials and Methods 2.1. Materials The selenium powders (Se, 99.9%), polyvinylpyrrolidone (PVP, Mw≈10000), iron chloride (FeCl3, Mw=162.2), ethylene glycol (EG, ≥99.0%) and pyrrole (99.0%) were

purchased

from

Shanghai

Macklin

Biochemical

Co.,

Ltd.

Sodium

terahydroborate (NaBH4, 98.0%) and bismuth nitrate pentahydrate (Bi(NO3)3·5H2O, ≥99.0%) were obtained from Adamas reagent Co., Ltd. Zinc phthalocyanine (C32H16N6Zn, >95.0%) was obtained from Tokyo chemical industry Co., Ltd. Hyaluronic acid (Mw≈10000, >95%) was purchased from Dalian meilun biological technology Co., Ltd. Dimethyl sulfoxide (DMSO, ≥99.0%) was purchased from Rionlin reagent Co., Ltd. All chemicals were of analytical grade and used without further purification. 2.2. Synthesis of Bi2Se3 Bi2Se3 core was synthesized via a simple solution based method. Mixing NaBH4 (2mmol, 0.074g) with Se powders (1 mmol, 0.079g) without oxygen to gain the NaHSe aqueous solution. Sealed the transparent solution for the following step. The Bi2Se3 nanodish was produced by the reaction between Bi(NO3)3·5H2O and the NaHSe solution as shown in the following. Poured 20ml PVP (0.5g PVP, 20mL EG) solution into 100mL round-bottom flask, then with magnetic stirring added the Bi(NO3)3·5H2O (0.226g Bi(NO3)3·5H2O, 12.5mL EG) solution at normal temperature. Then the mixed liquor heated to 160oC under N2. The previous synthesized NaHSe (free of oxygen) solution was injected rapidly into the mixture. Before down to room

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temperature, the reaction continued for 10 minutes. Precipitated outcomes through centrifuging and washing. 2.3. Synthesis of Bi2Se3@HA-doped PPy The Bi2Se3@HA-doped PPy was synthesized by one-pot method. In brief, Dissolve HA (400mg) in 20mL of DI water. Dissolve Bi2Se3 (0.0165g) and PVP (0.5g) in DI water (12.5mL), then added pyrrole (65µL). After 10 min, rapidly added 0.5mL of iron (III) chloride hexahydrate (0.75g/mL) to the reaction mixture. Added predissolved 20mL of HA solution in 2 min. After incubation for 9h, use a membrane dialysis (MWCO: 13kDa) to purify the solution for 2 days. Remove the large sized precipitate via centrifugation (12,000rpm, 2 min). Obtain the black powder by freeze drying. 2.4. ZnPc loading and releasing For ZnPc loading, BPH (2mg in 4mL DMSO) were mixed with ZnPc (0.5mg in 0.5mL DMSO). After stirring for 24h at room temperature, excess unloaded ZnPc was filtered (100 kDa Millipore filter) and rinsed with water. To verified drug release kinetics, Packaged 1mL BPHZn in a dialysis bag (MWCO: 13kDa) then put in 19mL of PBS at pH 7.4. At different time point, collected 2mL receptor solution and measure the concentrations of released drugs using the absorbance spectromete, then poured back to 20mL. To determine the NIR laser triggered ZnPc release kinetics, an 808 nm diodepumped solid-state laser system was employed at different time points in this work. 2.5. Measurement of photothermal performance Various samples (0.5mL) with the same concentration of BPH or ZnPc and BPHZn aqueous dispersions (0.5mL) with different concentration were irradiated 5 min with 808nm laser at 1.2W/cm2, respectively. Measured the temperature rise mediated by NIR laser through monitoring temperature of BPHZn nanoparticles dispersions in DI water at various concentrations (0, 25, 50, 70, 100µg/mL) irradiated by an NIR laser (808 nm, 1.2W/cm2). Temperatures were collected at designed time points. Real time thermal images of diverse prescription (0.5mL) were recorded as well. To measure photothermal conversion efficiency (η), recorded the change in 8


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temperature of the BPHZn dispersion under continuous 808nm laser with 1.2 W/cm2 as a function of time after the dispersion arrived a stable temperature. The value of η was gained through the below formula: η=

hS (TMax − TSurr ) − QS × 100% I (1 − 10 − A808 )

2.6. Detection of singlet oxygen 1,3-Diphenyl isobenzofuran (DPBF) was utilized to test the generation of singlet oxygen generation by BPHZn. Mixed a certain amount of BPHZn with DPBF (10µM) in acetonitrile. This mixture solution was then irradiated by NIR laser (670nm, 1.5W/cm2). 410nm absorbance was collected by a UV-vis spectrophotometer at different time points (44). 2.7. Cellular experiments We used MTT assay to test the photo-cytotoxicity using 4T1 cells (5×104/well) which were incubated in 96-wells for 24 h in an incubator. When the cells grew about 80% confluence, discarded the culture and washed cells(45). Added various formulations (ZnPc, BPH, BPHZn) at different concentrations into wells then incubated for 24 h after changing the cell culture, respectively. The cells in ZnPc wells, BPH wells and BPHZn wells were irradiated by 670nm laser (1.5W/cm2), 808nm laser (1.2W/cm2) and 808/670nm laser for 5min at 6h, respectively. Following that, added MTT solution (5 mg/mL, 20µL) to wells for further 4h incubation. Finally, in order to dissolve formazan crystals, we added 200µL DMSO into wells then measured the absorbance at 490 nm. We then use AO/PI co-stained study to observe live or dead cells respectively. We seeded the 4T1 cells (5×105/well) in CLSM culture dishes then incubated in PBS and different formulations (ZnPc, BPH, BPHZn). After 6h, ZnPc, BPH and BPHZn treated culture dishes were dealt with 670nm laser (1.5W/cm2), 808nm laser (1.2W/cm2), and 808/670nm laser with further incubated until 24h, respectively . After removing the culture medium, AO (10ng/mL, 1mL) and PI (10ng/ mL, 1mL) were added into culture dishes to incubated for 20min and 30min, respectively. Then, washed all dishes three times with PBS (pH 7.4) and observed through CLSM. 9



For cellular uptake studies of BPHZn nanodish complex, seeded 4T1 cells (1×105cells/well) into the CLSM culture dishes. After 12h incubation, various formulations with ZnPc (10µg/mL) were added and 2h later treated with 670nm laser irradiation (1.5W/cm2, 2min). Washed cells three times with PBS then fixed with 4% paraformaldehyde solution for 20 minutes after further incubation. Subsequently, the nuclei were stained with DAPI (10µg/mL) for 15 minutes. Lastly, washed the cells with PBS several times to remove excess fluorescent dyes then were captured using CLSM(46). 2’, 7’-dichlorofluorescein diacetate (DCFH-DA), a kind of ROS-sensitive probe was selected to test ROS generation. Once DCFH-DA entered the cells, it could be converted into DCFH (a nonfluorescent polar derivative) by intracellular esterase. Then ROS could oxidize DCFH rapidly, producing green fluorescent 2’, 7’-dichlorofluorescein (DCF).(47) Seeded 4T1 cells at the density of 1 × 106cells/well into CLSM culture dishes. After the different treatments, all groups were incubated with DCFH-DA. The intracellular ROS generation was visualized by using a confocal laser scanning microscope. ROS quantification was collected immediately by a multimode plate reader(48). 2.8. In vivo triple-modal imaging For FL imaging in vivo, 200 µL of free ZnPc or BPHZn nanoparticles (equivalent ZnPc 100 µM) were intravenously injected into 4T1 tumor-bearing Balb/c nude mice tail vein. After injection, acquired the time-dependent fluorescence images by ex/in vivo imaging system. After imaging in vivo, sacrificed the mice. Collected tumors and major organs for ex vivo imaging and quantitative biodistribution analysis. For in vivo PA imaging, 4T1 tumor-bearing Balb/c nude mice were intravenously injected through the tail vein with 200 µL of BPHZn nanoparticles with the same dose of 20 mg/kg. After injection, recorded PA images in the tumor sites at 0, 3, 5, 8 and 24 h. Determined the PA signal intensity via ImageJ. For CT imaging in vivo, 200µL of BPHZn nanoparticles (8mg/mL) were intratumor injected into each 4T1 tumor-bearing Balb/c nude mouse. Next, the mouse 10


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was set on a small X-ray CT for imaging at different time intervals (0 and 8h). The filtered back projection method was carried out to reconstruct images to obtain reconstruction images. And the Amira 4.1.2 system was used to analyze the reconstruction images. Following shows the main parameters: effective pixel size, 50µm; tube voltage, 80kV; field of view, 1024pixels × 1024pixels; tube current, 270µA. 2.9. In vivo combined therapy When the tumor size reached 200mm3, randomly divided the mice into five groups and i.v. administered with the following treatments: PBS only; PBS and exposed to the laser (PBS + 808nm); ZnPc and exposed to the laser (ZnPc + 670nm); BPH and exposed to the laser (BPH + 808nm); BPHZn and exposed to the laser (BPHZn + 808/670nm). Measured the tumor volumes every two days for 14 days and then calculated with: V = AB2/2, where A represent for maximum diameters of tumors and B represented the minimum diameters of tumors.(48) Body weights and survival curves were also recorded after the treatments. At day 14, removed tumors, together with major organs, fixed with 4% formalin then processed for hematoxylin and eosin (H&E) staining studies for pathological features. 2.10. Data Analysis All of the data were shown as the mean ± standard deviation (SD), and the different results of different treated groups were subjected to paired two-tailed Student’s t-test. The probability level of 95% (p < 0.05) was considered to be significantly different.

3. Results and Discussion 3.1. Preparation and characterization The Bi2Se3 nanodish was synthesised via a modified solution-based way utilizing PVP as surfactant.(26) As Scheme 1A shown, the Bi2Se3 core was sequentially sealed in a hyaluronic acid doped-polypyrrole (HA-doped PPy) shell to synthesize BPH by one-pot method. Next, added ZnPc into BPH nanoparticles solution and shook at normal temperature to form BPHZn. More details could be found in the Materials and

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Methods. In previously reported solution-based methods synthesizing Bi2Se3, Na2SeO3, which was normally required as the Se source, was reported to be toxic. On the contrary, Se powders and NaHSe used here were safer. BPH nanodish complex was synthesized through a one-pot way using Bi2Se3, pyrrole, FeCl3, hyaluronic acid together with DI water. The morphology of Bi2Se3 and BPHZn nanoparticles were demonstrated by transmission electron microscopy (TEM, Figure 1A, B and Figure 1D, E). Based on TEM images, the Bi2Se3 cores showed clear hexagon shape. We also characterized the Bi2Se3 nanodish complex by powder X-ray diffraction (XRD). As displayed in Figure 1C, indexed all the peaks to the rhombohedral phase of Bi2Se3. (49) In addition, the Bi2Se3 nanodishes were characterized by Raman scattering (Figure 1F). The Eg2 and A1g2 modes were observed at 120 and 162 cm−1, respectively. Based on the TEM imaging results, after coating HA-doped PPy the particle size was increased from ∼83nm for Bi2Se3 to ∼108nm for BPHZn nanodish complex (Figure 1G). The coated nanoparticles showed highly uniform, exhibiting low polydispersity index of Bi2Se3 (0.268) and BPHZn (0.110) measured by dynamic light scattering. Additionally, the zeta potential (ζ) varies after coating as well, exhibiting a negative charge (-17.3 mV), a positive potential (+3.7 mV) and a negative charge (-2.97 mV) for the naked Bi2Se3, BPH nanoparticles and BPHZn, respectively (Figure 1H). UV - vis adsorption in 300-900nm was also observed for Bi2Se3 and BPH. As Figure 1I demonstrated, the peak around 690 nm for BPHZn nanoparticles suggested the loading of ZnPc, which was further tested by fluorescence emission spectra (Figure 2A). Since that Bi2Se3, PPy and HA had no fluorescence emission, the peak of BPHZn nanoparticles at 690 nm was most probably attributed to ZnPc. As mentioned above, PPy nanoparticles had immense absorption coefficient with a broad peak, therefore through fluorescence resonance energy transfer (FRET) from the excited ZnPc to PPy nanoparticles the fluorescence of ZnPc was expected to be quenched when it near the PPy. As hypothesized, ZnPc fluorescence intensity largely decreased when loaded onto the BPH compared to free ZnPc (Figure 2A). The loading efficiency and capacity of ZnPc was measured to be 69.2% and 13.84% respectively, 12


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which was assessed by subtracting the redundant ZnPc in filtrate from the initial amount, and calculated based on the established ultraviolet absorption calibration curve. In addition, the BPHZn nanoparticles showed remarkable stability and good dispersion in water compared to naked Bi2Se3, which was seen in the photos of different formulations in day 1 and day 7 (Figure 2B, C). Similar to previous reports,(19) naked Bi2Se3 nanoparticles in our work were also not so stable, showing little sediment of the sample at the bottom of the bottle, color changing from black to dark brown after 7 days (water solution, room temperature). On the contrary, the appearance of BPH and BPHZn nanoparticles remained unchanged, providing proof of surface properties and the stability modified formulations boding well for biomedical applications. Combined the additional bio-imaging ability and therapy agents were highly wanted in imaging-guided therapy. Figure 1H displayed the photoacoustic images of BPHZn nanoparticles at Bi2Se3 concentrations between 0ppm and 250ppm. A linear relationship of Y = 20.55 + 4.72 X between the Bi2Se3 concentration and the PA signal intensity could be observed (R2 = 0.996). The PA conversion ability of the Bi2Se3 nanodish was much better than that of commonly used photothermal agents. Due to Bi element’s high X-ray attenuation coefficient, the viability of nanoparticles for CT imaging was evaluated at diverse nanoparticles concentrations in vitro. CT signal intensity gradually became stronger with the increasing nanoparticles concentrations (Bi concentrations: from 0 to 45.6 mM). We then calculated and organized CT value at diverse concentration (Figure 2E), exhibiting linear-increase with the concentration. The enhanced CT contrast efficacy of BPHZn nanoparticles was hopeful to decrease the potential toxicity and complications induced by high contrast agents dose during clinical applications. Furthermore, the NIR laser irradiation triggered PS release behavior from BPHZn was investigated in vitro. As illustrated in Figure 2F, without 670nm laser, the released ZnPc from the nanoparticles displayed only 10.7% and 13.1% release by 8h and 24h, respectively. While the ZnPc release from the nanoparticles under the 670nm 13



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laser irradiation reached to 38.1% during the first 8 h. At 24h, total ZnPc release amount was enhanced to 69.0%, revealing that the release profile can be well controlled and improved by laser irradiation. 3.2. In vitro PTT and PDT effect The photothermal conversion capability of BPHZn nanoparticles was verified by observing temperature rise with 670nm NIR laser at 1.2W/cm2. As depicted in Figure 3A, temperature was barely changed (no more than 6 oC) under irradiation in DI water group. On the contrary, Bi2Se3, BPH and BPHZn suspensions (100 µg mL-1) groups’ temperature were strikingly increased to 52.9, 58.2 and 59.1 oC over a 5 min period, respectively. As illustrated in Figure 3b, temperature rise followed a time- and concentration-dependent way. In particular, at 100µg/mL, temperature could reach to 43.0 oC under irradiation less than 2 min, which was the critical temperature asked for inducing cancer cells’ death.(50,51) The photothermal heating effects were measured by irradiating the aqueous solution under a low power density for 10 min (1.2W/cm2) under 670nm laser irradiation near the plasmon band. Adopting a previously reported method,(52) the photothermal conversion efficiency of the BPHZn nanodish complex was determined 36.35%, that was obviously better than that of common used photothermal agents. To further tested the photothermal stability of BPHZn nanoparticles, we performed four laser on/off cycles, irradiated the BPHZn nanoparticles dispersion via the NIR laser for 5 min (Laser on), then after Laser off cool to room temperature. As showm in Figure S1, the same elevated temperature from different cycles showed good photothermal stability since no significant decrease could be observed during temperature elevation. All of the results demonstrated that the BPHZn nanoparticles had good photothermal conversion efficiencysa well as excellent photothermal stability. To test producing cytotoxic ROS ability of BPHZn nanoparticles with 670nm laser

irradiation,

we

carried

out

a

chemical

method

by

using

the

1,3-diphenylisobenzofuran (DPBF) as an acceptor of ROS.(53) Figure 3F showed a significant decrease in the absorbance of DPBF after the optimal preparation exposed 14


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to the laser irradiation, indicating the generation of ROS. What’s more, the ROS generation of BPHZn was much lower and slower than that of free ZnPc under the same conditions, which could be attributed to the loss of effective energy absorbed by the BPH performing PTT effect in the mean time. 3.3. In vitro cellular experiments Toxicity of different preparations to 4T1 cells under diverse conditions was investigated via MTT assay. As Figure 4A depicted, without laser irradiation, cell viability of the different samples were all more than 90% even at the highest concentration (BPH 200µg/mL and ZnPc 10µg/mL). When exposed to laser, however, all treatments showed concentration-dependent tumor cell killing abilities (Figure 4B). Moreover, compared with free ZnPc or BPH alone treated group, the optimal formulation BPHZn experienced the highest therapeutic efficacy. The results were also verified by fluorescence co-staining method utilizing Calcein AM (green) and PI (red) in Figure 4C. Synergistic group of BPHZn exhibited the strongest red color, suggesting the best anti-tumor efficacy compared with the other therapy groups. We also evaluated the cellular uptake and intracellular ROS generation of multiple samples with determined laser conditions on 4T1 cells at an identical ZnPc concentration (10µg/mL). As Figure 4D shown, majority of BPHZn nanodish complex was distributed in the cytoplasm after 4 h incubation. What’s more, we could observe an enhanced ZnPc fluorescence in the cell under a short time laser irradiation (808nm, 1.2W/cm, 2min), suggesting dequenched ZnPc release from the formulation. Next, we evaluated the intracellular ROS generation by BPHZn nanoparticles in 4T1 cells through the confocal microscopy observation (Figure 4D). ROS was usually considered to oxidize proteins and DNA in cells, inducing serious cytotoxicity via initiating pro-apoptotic cellular signal and stimulating inflammation.(54,55) In our work, 2,7-dichlorofluorescein diacetate (DCF-DA) was employed as the intracellular ROS-production indicator, because nonfluorescent DCF-DA can be oxidized to fluorescent DCF (green) in presence of ROS. After incubated 4T1 cells with BPHZn nanoparticles and excited by combined 808/670nm laser irradiation, obvious green fluorescence was observed (Figure 4D), suggesting intracellular ROS generation. In 15



contrast, cells treated with free ZnPc+670nm laser irradiation showed negligible green fluorescence, which may due to the limited uptake of free ZnPc. The results indicated the promising ROS production ability of BPHZn nanoparticles inside the tumor cells. 3.4. In vivo PA/FL/CT images The triple-modal imaging capacity of BPHZn nanodish nanocomplex was evaluated. After intravenous (i.v.) injection of BPHZn or free ZnPc, fluorescence signals were respectively gathered at 0, 3, 5, 8, and 24 h time intervals as shown in Figure 5A, B. In BPHZn treated group, at the early stages of post-injection, we could see fluorescence signal distributed widely all through the body, likely because of the high nanoparticle concentration in blood vessels. Then, tumor fluorescence signals increased and at 8 h post-injection reached to the peak value, demonstrating sustained accumulation in tumor as well as confirming the appropriate time to conduct the lasers. On the contrary, mice injected with free ZnPc showed negligible tumor contrast fluorescence images(Figure 5A, top panel). These results implied that BPHZn nanoparticles could circulate in the blood flow and target tumors rapidly. To clearly observe the biodistribution of BPHZn nanoparticles, major organs and tumors were resect after 24h injection. Obviously, the optimal formulations were mainly distributed in the tumor, free ZnPc was primary distributed in liver (Figure 5A, B bottom panel). Bio-distribution semi-quantitative analyses in different tissues further confirmed the high tumor retention of BPHZn nanoparticles in comparison of other major organs (Figure 5C), which might be ascribed to the dual-targeting ability (CD44 binding and enhanced permeability and retention effect) of BPHZn nanodish complex. The capacity of BPHZn nanodish complex for in vivo PA imagings was demonstrated (Figure 5D). The PA signal of the optimal formulation remarkably increased over time after i.v. injection, indicating effective tumor retention through both passive and active targeting pathways. In good agreement with the fluorescence imaging results, the PA signal intensity showed at 8h post-injection BPHZn nanodish complex reached the maximal tumor uptake quantity (Figure 5E). To examine the CT contrast performance of BPHZn nanodish complex, we intratumoral injected 4T1 16


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tumor-bearing with the BPHZn nanodish complex for CT imaging of the whole body 8h post injection (Figure 5F and G). Marked tumor CT signal was found, in comparison with pre-injection images, suggesting the adequate tumor retention of nanoparticles. Additionally, Hounsfield units (HU) numbers, presented CT contrast effect, rose from 121.4 ± 22.9 (pre-injection) to 236.3 ± 39.4 (post-injection) (Figure 5H). These results demonstrated that the BPHZn nanoparticles experienced notable trimodal NIRF/PA/CT imaging capabilities and obvious tumor-homing effect. 3.5. Combined therapy in vivo To verified combined PTT and PDT effect, we also conducted in vivo experiments. 4T1 tumor-bearing mice were divided into five groups randomly: (1) PBS as the control; (2) PBS + 808nm; (3) BPH + 808nm; (4) free ZnPc + 670nm; (5) BPHZn + 808/670nm. After intravenous injection with 100µL PBS, BPH, free ZnPc, or BPHZn (5.0mg/mL, containing the equal ZnPc quantity), radiated the tumors with 808nm laser (1.2W/cm, 5min) and monitored temperature change. Figure 6 demonstrated that infrared thermal imaging with high-contrast could be realized by BPH and BPHZn in vivo once again. For BPH or BPHZn treated groups, tumor sites became brighter as increasing irradiation time, demonstrating the efficient tumor accumulation. There was minor increase of tumor temperature for the free ZnPc- and PBS- treated group (∆Tfree ZnPc ~ 9.7 °C, ∆TPBS ~4.9 °C) after 5 min irradiation. On the contrary, for the BPH and BPHZn groups, tumor sites temperature dramatically up to 49−53 °C during irradiation, sufficiently induced hyperthermia. We also particularly collected the tumor change of the optimal formulation group after treatment. Figure 7A showed that the tumor was decreased largely and left previous tumor sites with small scars. Measured tumor size every 2 days and plotted changes of tumor volume after treatment. In free ZnPc and PBS control group, the tumor grew quickly in the following days (Figure 7C). These results indicated that treated with such low dose of the free ZnPc seldom performed antitumor capabilities. In the BPH + 808nm group treated group, in the first few days the tumors were inhibited but afterward grew uncontrollably. In comparison of the control group, the tumor inhibition ratio of the PTT group was ~68.0%. Notably, in BPHZn under laser irradiation treated group, 17



tumor inhibition ratio was up to ~96.4% during the whole experimental period. As demonstrated in Figure 7B, average tumor weight in combined therapy treated group was the lightest among all the groups. Noticeably, BPHZn + 808/670nm treated group showed a 100% survival after 36 days. In contrast, mice of other treated groups had lower survival or even dead out owing to the extensive tumor burden (Figure 7E). These in vivo results demonstrated that, in comparison with either PDT or PTT alone treated group, combined therapeutic of PDT/PTT showed the considerably higher tumor inhibition efficacy. 3.6. In vivo toxicity Next we verified potential toxic of nanoparticles in vivo. We measuring the body weights and monitoring the behaviors of mice after i.v. injection with the BPHZn. There is no remarkable unnatural phenomenon in eating, drinking, neurological status, grooming, exploratory behavior, activity, or urination occurred while the experimental time. Moreover, as demonstrated in Figure 7D, no remarkable changes in mice body weight for all treatments, concluding no/low in vivo systemic toxicity of the nanoparticles. Besides, after the treatments sacrificed the mice. Collected major organs for histology studies. No notable inflammatory lesion or damage were found in all of the organs in Figure 6F, further verifying no remarkable in vivo toxicity under our tested dose at least. All of these in vivo results demonstrated that the BPHZn nanoparticles is a potential smart drug nanocarrier for high efficient PTT/PDT without significant toxicity at administrated dose, exhibiting excellent competent for biomedicine applications.

Conclusion In this study, we designed a multi-functional nanoplatform based on Bi2Se3@HA-doped PPy/ZnPc nanodish complex for multi-modal imaging-guided photothermal-photodynamic combined antitumor therapy. We utilized HA-doped PPy as a smart nanoplatform to effective functionalize Bi2Se3 nanomaterials, enable Bi2Se3 to possess high drug loading abilities, remarkable stabilities and tumor-targeted

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abilities. BPHZn nanodish complex with high-contrast CT, PA and FL imaging, could be utilized for excellent photothermal/photodynamic (PTT/PDT) combined therapy for tumor inhibition and eradication. The PTT/PDT mediated by the nanoparticles is considerably more effective than PTT or PDT alone, realizing 1+1>2 performance. Moreover, our work finds a new way to function 2D nanomaterials sharing similar properties and structures with Bi2Se3 for biomedical usage.

Acknowledgement This work was supported by the Peiyang Young Talent Fund of Tianjin University (1701), National Natural Science Foundation of China (81503016), National Basic Research Project(973 Program) of China (2014CB932200), and Application Foundation and Cutting-edge Technologies Research Project of Tianjin (Young Program) (15JCQNJC13800). Supporting Information Temperature elevation of BPHZn nanodish complex over 4 irradiation cycle, Bi and Se concentrations in major organs at different day post-injection of BPHZn, residual amounts and corresponding residual ratios of Bi and Se plotted versus time post-injection of BPHZn.

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Scheme 1. Schematic illustration of the (A) preparation produces and (B) working mechanism of BPHZn nanoparticles.

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I

Figure 1. Formulation characterization. TEM images of (A) Bi2Se3 nanodishes and (D) Bi2Se3@HA-doped PPy/ZnPc (BPHZn) nanoparticles. HRTEM images of (B) Bi2Se3 nanodishes and (E) Bi2Se3@HA-doped PPy/ZnPc (BPHZn) nanoparticles. (C) XRD spectrum of Bi2Se3. (F) Raman spectrum of Bi2Se3. (G) Size distribution of Bi2Se3 nanodishes and Bi2Se3@HA-doped PPy/ZnPc (BPHZn) nanoparticles. (H) Zeta potential of Bi2Se3 nanodishes and Bi2Se3@HA-doped PPy/ZnPc (BPHZn) nanoparticles. (I) UV-vis-NIR spectra of Bi2Se3, Bi2Se3@HA-doped PPy (BPH) and Bi2Se3@HA-doped PPy/ZnPc (BPHZn).

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Figure

2.

Formulation

characterization.

(A)

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Fluorescence

spectra

of

Bi2Se3@HA-doped PPy (BPH), ZnPc and Bi2Se3@HA-doped PPy/ZnPc (BPHZn). Digital photos of nanoparticles in (B) day1 and (C) day7. (D) In vitro PA images of Bi2Se3@HA-doped PPy/ZnPc (BPHZn) at different concentrations and linear fitting of the PA signal intensities as functions of Bi2Se3 concentration. (R2=0.996) (E) In vitro CT contrast images of Bi2Se3@HA-doped PPy/ZnPc (BPHZn) at different concentrations and linear fitting of the CT value as a function of Bi concentration (R2 = 0.989). (F) ZnPc release profiles of Bi2Se3@HA-doped PPy/ZnPc (BPHZn) nanoparticles at 37 °C or with laser irradiation (1.2 W/cm2) for 3 min.

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Figure 3. In vitro photo properties of nanoparticles. (A) Temperature increase of different formulations (100 µg mL-1, 500 µL) under laser irradiation (670nm, 1.2 W/cm2) as a function of time (0−300 s). (B) Temperature increase of Bi2Se3@HA-doped PPy/ZnPc (BPHZn) at varying concentrations (0−100 µg mL-1, 500 µL) under laser irradiation (670nm, 1.2 W/cm2) as a function of time (0−300 s). (C) Infrared thermal images of different formulations droplet (100 µg mL-1) irradiated with a 670nm (1.2 W cm-1) laser for 300 s. (D) Photothermal effect of the irradiation of the aqueous solution of the Bi2Se3@HA-doped PPy/ZnPc (BPHZn) nanoparticles with the NIR laser (670nm, 1.2 W cm-1), in which the irradiation lasted for 600 s and then the laser was shut off. (E) Linear time data versus −ln(θ) obtained from the cooling period of (D). The time constant (τs) for heat transfer of the system is determined to be 265.57 s. (F) ROS generation under laser irradiation (670nm, 1.2 Wcm-2).

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Figure 4. Cell viability of 4TI cells incubated with various concentrations of free ZnPc, BPH and BPHZn without (A) or with (B) NIR laser irradiation. Data are presented as means ± SD (n = 3). (C) CLSM images of 4T1 cells treated with PBS, free ZnPc, BPH and BPHZn with NIR laser irradiation. Viable cells were stained green with calcein-AM, and dead/later apoptosis cells were floating and eluted or stained red with PI. Scale bar: 200 µm. (D) CLSM images of 4T1 cells treated with PBS, free ZnPc + 670nm, BPHZn + 808nm and BPHZn + 808/670nm for ROS detection.Scale bar:25µm.

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Figure 5. In vivo FL/PA/CT images. (A) Fluorescence images of tumor bearing mice at different time points after administration of free ZnPc and (B) BPHZn nanoparticles; the bottom panel shows the ex vivo images examined at 24 h post-injection. (C) Relative fluorescence intensity of ZnPc in major organs induced by laser irradiation after administration of free ZnPc and BPHZn nanoparticles injection for 24 h, **p < 0.01. (D) Ultrasound (US) images and PA images of 4T1 tumor-bearing mice after intravenously injected BPHZn nanoparticles at different time points. (E) Corresponding photoacoustic signal intensity of the BPHZn nanoparticles in the tumor at different time points. (F) In vivo 2D and (G) 3D CT images of mice before (0h) and 8 h after intravenously injected with BPHZn nanoparticles. (H) Corresponding HU value of BPHZn nanoparticles in the tumor sites before (0h) and 8 h after injected the BPHZn nanoparticles.(**p

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