Multifunctional Bi@PPy-PEG Core-Shell Nanohybrids for Dual-Modal

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Multifunctional Bi@PPy-PEG Core-Shell Nanohybrids for Dual-Modal Imaging and Photothermal Therapy Sisi Yang, Zhenglin Li, Yuanlin Wang, Xuelei Fan, Zhaohua Miao, Ying Hu, Zhuo Li, Ye Sun, Flemming Besenbacher, and Miao Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17838 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 23, 2017

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ACS Applied Materials & Interfaces

Multifunctional Bi@PPy-PEG Core-Shell Nanohybrids for Dual-Modal Imaging and Photothermal Therapy Sisi Yang,†,# Zhenglin Li,†,# Yuanlin Wang,†,# Xuelei Fan,† Zhaohua Miao,∫ Ying Hu,║ Zhuo Li,† Ye Sun,*,‡ Flemming Besenbacher,§ and Miao Yu*,† †

State Key Laboratory of Urban Water Resource and Environment, School of Chemical

Engineering and Technology, Harbin Institute of Technology, Harbin 150001, China ‡

Condensed Matter Science and Technology Institute, Harbin Institute of Technology, Harbin

150001, China §

Interdisciplinary Nanoscience Center (iNANO) and Department of Physics and Astronomy,

Aarhus University, Aarhus 8000, Denmark ∫

School of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150001,

China ║

School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, China

* Corresponding authors. Email: [email protected] (M. Yu) and [email protected] (Y. Sun) #

These authors contributed equally to this work.

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ABSTRACT High-performance theranostic nanoagents which integrate multi-modal imaging and photothermal therapy for clinical anticancer treatment are highly desired. Herein, we report the synthesis and bioapplication of a multifunctional theranostic nanoagent based on polyethylene glycol (PEG)modified polypyrrole (PPy)-coated bismuth (Bi) nanohybrids (referred as Bi@PPy-PEG NHs) for X-ray computed tomography/photoacoustic (CT/PA) dual-modal imaging and photothermal therapy (PTT). The obtained Bi@PPy-PEG NHs have a distinct core-shell structure with the metallic Bi nanoparticle as the inner core, and the PPy-PEG layer as the shell. The Bi@PPy-PEG NHs show excellent physiological stability and compatibility, without noticeable cytotoxicity. Importantly, the NHs exhibit strong NIR absorbance and remarkable photothermal conversion capability and conversion stability, with the photothermal conversion efficiency as high as ∼ 46.3%. Thanking to the strong PTT effect, highly effective photothermal ablation on cancer cells has been achieved both in vitro and in vivo. Furthermore, a high-contrast in vitro and in vivo CT/PA dual-modal imaging has been realized, showing great potential to provide comprehensive diagnosis information for antitumor treatment. In particular, the CT enhancement efficiency of the NHs is of ~ 14.4 HU·mM-1, which is ~3.7 fold as that of clinically-used iohexol, Therefore, our work highlights the potential of using such core-cell Bi@PPy-PEG NHs as a versatile theranostic nanoplatform for cancer imaging and therapy.

Keywords: bismuth nanoparticles, photothermal therapy, computed tomography imaging, photoacoustic imaging, core-shell structure


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1. INTRODUCTION Imaging-guided photothermal therapy (PTT) has sparked tremendous interests in recent years due to its considerable advantages, including spatial controllability guided by on-site diagnosis, realtime monitoring on therapeutic effects, remote and targeting treatment without damage to surrounding tissues.1-3 Albeit the huge effort devoted in this field, exploiting high-performance theranostic agents remains challenging. Especially, multiple imaging modes are indispensable in most cases. For instance, photothermal agents (PTAs) may hold potentials for photoacoustic (PA) imaging, which integrate optical excitation with ultrasonic detection based on the PA effect induced by the near-infrared (NIR) light absorption and subsequent thermal expansion, and has become particularly popular lately due to its high sensitivity and real-time monitoring on soft tissues.4,5 However, some inherent defects, such as the limited light penetration, low signal response from tumor tissues, and high background interference of PA imaging require additional imaging mode(s) to remedy its drawbacks.6,7 Encouragingly, unlike organic PTAs, e.g. polypyrrole (PPy) and indocyanine green (ICG), some inorganic PTAs developed recently indeed possess a second inherent imaging mode, such as X-ray computed tomography (CT) from metal-based nanostructures (e.g. Au nanocages, Ag nanodots, Pd nanosheets),8-10 magnetic resonance imaging (MRI) based on Co-doped PTAs or Fe3O4 composites,11 or fluorescence imaging of carbon nanodots.12 However, quite a few concerns are also raised accordingly. Firstly, the long-term safety of most inorganic PTAs remains uncertain. And the potential toxicity and side effects can be further exacerbated by the large dose demanded by the low photothermal conversion efficiency or/and deficient imaging contrast. Moreover, the unwanted degradation of many PTAs in the physiological environments in vivo can severely decrease or even completely deprive their NIR absorption and photothermal conversion capability before reaching the tumor areas. Therefore, it is of paramount importance to


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explore biocompatible and stable nanoagents simultaneously owning powerful multimodal imaging functions and high PTT efficacy. Bismuth (Bi) compound nanomaterials have attracted intense attention of the biomedical society in recent years.13-17 In our earlier work, we have demonstrated that a series of Bi-containing nanoagents, including PEGylated Bi2S3 nanourchins,18 polymer-coated Bi2Se3 nanoplates,19 and highly porous Bi2Se3 spherical-sponges,20 can perform as potent PTAs for PTT or thermochemotherapy, integrating with the high-contrast CT imaging. As the most convenient and widelyused diagnostic method in clinic, CT possesses high temporal and spatial resolution,5,21 which can make a perfect couple with PA imaging. The CT contrast at a certain mass concentration replies on the high atomic-number (Z) elements and their density in the agents. Bi is a typical high-Z element (I, 53; Ta, 73; Au, 79; Bi, 83) with a high X-ray attenuation coefficient (I, 1.94; Ta, 4.30; Au, 5.16; Bi, 5.74 cm2·kg-1 at 100 keV).22 Obviously, the Bi content hence the CT enhancement efficiency can be largely increased in pure Bi nanoparticles compared with the Bi compounds. Although the possibility of Bi nanoparticles for in vivo CT/PT imaging and PTT/radiotherapy has been demonstrated very lately,23-25 it remains intriguing and passionate to develop new Bi PTAs with superior imaging functions, photothermal conversion capability and higher stability. Herein, we develop a multifunctional theranostic nanoagent based on polyethylene glycol (PEG)-modified polypyrrole (PPy)-coated Bi nanohybrids (Bi@PPy-PEG NHs) and investigate their multimodal imaging and PTT performance. The Bi@PPy-PEG NHs are fabricated by utilizing pure metallic Bi nanoparticle as the inner core and the PPy layer self-assembled via a facile onestep chemical oxidation polymerization as the shell, followed by PEG modification (Scheme 1). Compared with the metallic Bi nanoparticles, the resultant core-shell Bi@PPy-PEG nanohybrids (NHs) possess excellent physiological stability and compatibility, without noticeable cytotoxicity.


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Importantly, the NHs exhibit an improved photothermal conversion capability and conversion stability upon repeated irradiation cycles. Especially, the photothermal conversion efficiency is significantly increased compared with that of the Bi core (∼46.3% vs. ~30.4%). Due to the strong PTT effect, highly effective photothermal ablation on cancer cells has been achieved in vitro/vivo after treated with the NHs plus NIR laser irradiation. Furthermore, a high-contrast CT/PA dualmodal imaging has been demonstrated both in vitro and in vivo, showing great potential to provide accurate diagnosis information for antitumor treatment. Especially, the CT enhancement value is as high as 14.4 HU·mM-1. Our work highlights the potentials of using such core-cell Bi@PPy-PEG NHs as a versatile theranostic nanoplatform for cancer imaging and therapy.

Scheme 1. Illustration of the synthesis and multifunction of the Bi@PPy-PEG NHs.


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2. EXPERIMENTAL SECTION 2.1. Materials. Bismuth nitrate pentahydrate (Bi(NO3)3·5H2O, 99%), D-(+)-glucose, 1,2propanediol (PPD, 99%), borane-morpholine complex (C4H9NO·BH3, ≧ 95%), pyrrole (Py, 99%), iron chloride hexahydrate (FeCl3·6H2O) were purchased from Aladdin (China). Polyvinyl alcohol 124 (PVA, GR) was purchased from Sinopharm (China). Dopamine-polyethylene glycol (DA-PEG, MW ≈ 2000) was provided by Xi’an Ruixi Biological Technology Co. Ltd. (China). Cell Counting Kit-8 (CCK-8), calcein acetoxymethyl ester (Calcein AM, > 90.0%), and propidium iodide (PI) were purchased from Dojindo Laboratories. All reagents were of analytical grade and used without further purification. Deionized (DI) water with a resistivity of ~ 18.2 MΩ cm was obtained from a Milli-Q Water Purification System and used in all the experiments. 2.2. Synthesis of Bismuth Nanoparticles. Bi(NO3)3·5H2O (485 mg), D-(+)-glucose (10.0 g) and PPD (88.0 mL) were mixed in a three-neck flask, then mechanically stirred at 80 ℃ until the solution became transparent. Subsequently, borane-morpholine (308 mg) dissolved in PPD (12.2 mL) was quickly added, and the mixture turned black immediately. After one-minute reaction, the product was allowed to cool to room temperature, then centrifuged (13000 rpm, 20 min) and washed with DI water for three times. 2.3. Synthesis of the Bi@PPy-PEG NHs. The NHs were synthesized by in-situ chemical oxidation polymerization of Py onto the Bi nanoparticles surface. Briefly, FeCl3 aqueous solution (20 mg·mL1

, 8.0 mL) and Bi nanoparticles dispersion (0.1 mg·mL-1, 20.0 mL) were consecutively added to

PVA solution (10 mg·mL-1, 60.0 mL) under vigorous stirring. 1 h later, Py (100 µL) was added to the mixture, and the reaction was kept at 5 °C for 20 h. The obtained Bi@PPy was centrifuged (12000 rpm, 10 min) and washed with DI water for three times to remove extra reagents. For PEG modification, the Bi@PPy (4.0 mg) was fully dispersed into DA-PEG solution (2 mg·mL-1, 2.0 mL)


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and then stirred overnight. The obtained product was centrifuged, washed with DI water, and finally dispersed in DI water before use. 2.4. Characterization. The morphology of the Bi nanoparticles and Bi@PPy-PEG NHs were characterized by transmission electron microscopy (TEM, Tecnai G20, FEI Co., USA). Optical absorption was measured by Ultraviolet−visible−near-infrared spectrophotometer (UV−vis−NIR, Evolution 300, Thermo Scientific, USA). A Fourier transform-infrared spectrometer (FT-IR, Thermo Scientific, Nicolet 6700 USA) was used to measure the FT-IR spectra. The crystallization nature of the samples was investigated by powder X-ray diffraction (XRD, Bruker Advanced D8 Discover, USA) with Cu Kα radiation. 2.5. Measurement of Photothermal Properties. To test the photothermal effect, the Bi@PPy-PEG NHs aqueous dispersions (1.0 mL) at different concentrations (0, 10, 50, 100, 150 µg·mL-1) were transferred to every quartz cuvette (total volume of 4.0 mL), then irradiated with an 808 nm laser (2.0 W·cm-2) for 10 min, and the temperature was recorded by a thermocouple probe submerged in the solution. Meanwhile, the system was photographed by an infrared thermal imaging camera every two minutes. To further calculate the photothermal conversion efficiency of the sample, DI water (1.0 mL) and the Bi@PPy-PEG NHs (100 µg·mL-1, 1.0 mL) dispersion were irradiated for 20 min and then cooled to room temperature. For photothermal stability assessment, the Bi@PPy-PEG NHs dispersion was repeatedly (5 times) irradiated for 3 min, following by naturally cooling for 12 min. The light absorbance was measured before and after the repeated cycles, respectively. 2.6. Cytotoxicity Study. Human umbilical vein endothelial cells (HUVEC) and murine breast cancer 4T1 cells were incubated in 96-well cell-culture plates (1 × 104 cells per well) for 24 h, and then treated by the Bi@PPy-PEG NHs dispersions at various concentrations (0, 10, 50, 100, 150, 200 µg·mL-1, 100 µL) for 24 h and 48 h, respectively. The cell viability was measured by the CCK-


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8 assay. A microplate absorbance reader (BIO-680, USA) was used to measure the absorbance at 450 nm. 2.7. In Vitro Photothermal Therapy Effect. In order to investigate the photothermal therapy effect of the Bi@PPy-PEG NHs on cancer cells, 4T1 cells were cultured in a 12-well cell-culture plate for 24 h, then incubated with the Bi@PPy-PEG NHs dispersion (100 µg·mL-1) for another 24 h. Subsequently, the cells were repeatedly washed to remove the free NHs in the incubation medium, and the medium was replaced with fresh medium. The cells after the treatments were irradiated by the 808 nm laser (2.0 W·cm-2) for 0 and 5 min. Washing three times with phosphate buffer saline (PBS), then stained with calcein-AM (2 µmol·L-1) and PI (3 µmol·L-1) for 30 min to observe the living and dead cells. To quantitatively evaluate the photothermal effect, the Bi@PPy-PEG NHs dispersions at gradient concentrations (0, 50, 100 µg·mL-1) were incubated with 4T1 cells for 24 h and then irradiated by the 808 nm laser. The cell viability was then examined by the CCK-8 assay. 2.8. In Vivo Photothermal Therapy Effect. Tumor-bearing BALB/c mice were divided into three groups (n=3). The mice were intravenously injected with the Bi@PPy-PEG NHs dispersions (10.0 mg·mL−1, 200 µL). 24 h later, the mice were treated with/without 808 nm laser (1.5 W·cm-2) for 15 min. During the irradiation, the tumor temperature was recorded using an IR thermal imager (Ti25, Fluke, USA). Afterwards, the length (a), width (b) of the tumor, and the weigh of the mice were measured every 2 day, respectively. The tumor volume was calculated as V = a × b2/2. Finally, the mice from different groups were sacrificed for necropsy, and the major tissues were harvested and fixed in 4% paraformaldehyde solution, embedded in paraffin, and cryo-sectioned into slices of 4 µm in thickness. The frozen slides were further stained with hematoxylin & eosin (H&E) and examined under an inverted fluorescence microscope (IX71, Olympus, Japan) with a digital camera.


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2.9. In Vivo Biodistribution. Tumor-bearing BALB/c mice (n=3) were intravenously injected with the Bi@PPy-PEG NHs dispersions (10.0 mg·mL−1, 200 µL in PBS). 24 h later, the major organs (including heart, liver, lung, kidney spleen and tumor) and the tumor were harvested and weighted. The Bi contents in these organs and the tumor were analyzed by inductively coupled plasma-mass spectroscopy (ICP-MS). 2.10. Hemolysis Assay. Diluted red blood cells (RBCs) suspension (0.1 mL) was added to Bi@PPy-PEG NHs dispersions (0.9 mL) at various concentrations (from 100 to 1000 µg·mL−1). RBCs incubated with DI water and PBS were used as the positive and negative control, respectively. After 24 h, all the samples were finally centrifuged at 15000 rpm for 10 min. The 577nm absorbance of the supernatant was measured by UV-vis-NIR spectroscopy. The hemolysis percent of RBCs was calculated by the following equation: percentage of hemolysis (%) = (As − An)/(Ap − An) × 100%, As, An and Ap are the absorbance of the sample, the negative and positive control groups, respectively. 2.11. In Vitro and In Vivo CT and PA Imaging. For in vitro CT imaging, the Bi@PPy-PEG NHs dispersions at various concentrations of Bi (0, 0.27, 0.53, 4.26, 8.51, 17.02 mmol·L-1, 1.0 mL) were scanned by a mice X-ray CT system (80 mA, 100 kV). The CT values (Hounsfield Unit, HU) were measured in the region of interest. For in vivo CT imaging, the Bi@PPy-PEG NHs dispersion (5.0 mg·mL-1, 100 µL) was intratumorally injected into the tumor-bearing BALB/c nude mouse, followed by anesthetizing with isoflurane for CT scanning under the mice X-ray CT system (80 mA, 100 kV, slice thickness of 0.625 mm). The resulting CT images were analyzed using Amira 4.1.2. As for in vitro PA imaging, the Bi@PPy-PEG NHs dispersions at various concentrations (0, 14, 56, 111, 223, 445 µg·mL-1, 1.0 mL) in centrifuge tubes were imaged by a PA tomography system (Endra Nexus 128, Ann Arbor, MI) under the excitation wavelength of 808 nm. The PA signals


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were quantitatively analyzed based on the region of interest. For in vivo PA imaging, tumor-bearing BALB/c nude mice were intratumorally injected with the Bi@PPy-PEG NHs suspension (100 µL, 2.0 mg·mL-1), and then scanned with the PA imaging system (Endra Nexus 128, Ann Arbor, MI). The corresponding PA image before injection was also taken as the control. All the experiments on animals were performed according to the protocols approved by the Institutional Animal Care and Use Committee.

3. RESULTS AND DISCUSSIONS 3.1. Synthesis and Characterization. The Bi nanoparticles were first synthesized by using D-(+)glucose saturated PPD as the solvent and borane-morpholine complex as the reducing agent. As shown in Figure 1a, the typical TEM image reveals that the as-grown Bi nanoparticles appeared as faceted and uniform nanocrystals with an average diameter of ~ 70 nm. Energy dispersive spectroscopy (EDS) and powder XRD analysis were carried out to study the chemical composition and crystallization nature. It was revealed that, except the C, O and Si elements from the substrate used for measurement, only intense Bi signals were detected in the EDS analysis (Figure 1b). All the diffraction peaks in the XRD pattern (Figure 1c) can be well-indexed to the standard hexagonal structure of Bi crystal (PDF card JCPDS 85-1330), and no additional peaks attributed to any other phases or components was observed. Consistent with the TEM results and EDS analysis, the results indicate the high crystallinity and purity of the Bi nanoparticles.


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Figure 1. (a) Typical TEM image, (b) EDS analysis and (c) XRD pattern of the Bi nanoparticles. (d) TEM image and (e) size distribution histogram of the Bi@PPy-PEG NHs. (f) FT-IR spectra of the Bi nanoparticles, PPy, Bi@PPy, DA-PEG and Bi@PPy-PEG NHs.

It was found in the experiments that, the bare Bi nanoparticles could be oxidized slowly and form the amorphous white precipitate over time when kept in water at room temperature (Figure S1). To improve the physiological stability and biocompatibility, the Bi@PPy-PEG NHs were then fabricated via a facile one-step in-situ chemical oxidative polymerization of Py onto the Bi nanoparticles surface by using PVA as the stabilizer and FeCl3·6H2O as the initiator. Dopamine (DA)-terminated PEG (Figure S2) was used for PEG coating, as DA can self-polymerize to form a polydopamine (PDA) film that is capable to firmly attach on almost any surface, thanking to the diphenol and amino groups.26-28 As shown in Figure 1d, the obtained Bi@PPy-PEG NHs had a distinct core-shell structure with the Bi nanopartile as the inner core and the PPy-PEG layer as the shell (thickness ~ 35 nm). The average size of the NHs was increased to ~ 151 nm (Figure 1e).


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Moreover, the high-resolution TEM (HRTEM) image of the Bi@PPy-PEG NHs (Figure S3) showed an inter-planar spacing of ∼0.33 nm, corresponding to that of Bi(012) planes and same to that of the NHs before PPy-PEG coating, confirming that the coating did not change the Bi core noticeably. The results of FT-IR spectra (Figure 1f) further confirmed the successful PPy-PEG coating. The peaks at 1550 cm-1 and 1640 cm-1 are corresponding to the stretching vibration of the pyrrole ring in PPy, and the peak at 1100 cm-1 are assigned to the stretching vibration of C-O-C in PEG.29,30 In contrast to the slow oxidation of the bare Bi nanoparticles, the relatively hermetic PPyPEG coating layer of the Bi@PPy-PEG NHs indeed resulted in much improved stability and antioxidative property, not only in water (Figure 2a), but also in acidic, neutral, or alkaline PBS (Figure S4), showing no detectable change when kept at room temperature for one month or longer. Moreover, the Bi@PPy-PEG NHs exhibited excellent dispersity and compatibility in various physiological solutions without any sign of macroscopic aggregation (Figure S5). Furthermore, the average hydrodynamic size of the NHs was measured using dynamic light scattering (DLS) before and after 9-day storage in various physiological mediums at room temperature (Figure S6). For the fresh samples, the measured sizes in DI water, PBS, and DMEM with 10% FBS were ∼143.5 nm, 153.1 nm, and 137.4 nm, respectively. After 9-day storage, the values were slightly increased to be ∼148.8 nm, 163.7 nm, 142.8 nm, respectively. All of these results indicate the high stability of the NHs in different physiological solutions. The high stability was further confirmed by the linearly increasing 808-nm absorbance with increased Bi@PPy-PEG NHs concentrations in both water and DMEM culture medium (Figure S7).

3.2. Photothermal Properties. The optical absorption property of the Bi@PPy-PEG NHs was studied by the UV–vis–NIR absorbance spectra, showing a broad and strong absorption in the entire


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measured range from 400 to 900 nm (Figure 2a). To investigate the photothermal conversion capacity, the Bi@PPy-PEG NHs dispersions at gradient concentrations (0, 10, 50, 100, 150 µg·mL−1, 1.0 mL) were added into every quartz cuvette of 4.0 mL, then irradiated by the 808 nm laser, respectively. The temperature variation was recorded by a thermocouple probe immersed in the dispersions every 1 s. As shown in Figure 2b, rapid temperature rise of the NHs dispersions was observed upon laser irradiation, following a concentration- and irradiation time-dependent manner. When the concentration of the Bi@PPy-PEG NHs dispersions was gradually increased from 10 to 150 µg·mL-1, the measured temperature elevation (∆T) was 20.3, 39.0, 54.6, and 59.5 °C upon 10min irradiation, respectively (Figure 2c). In sharp contrast, the temperature of pure DI water was only increased ~ 2.6 °C under the same irradiation. It should be pointed out that, at 150 µg·mL−1, the system temperature can be increased to 87.3 °C after 10-min irradiation, and to 50.0 °C after a rather short irradiation for less than 80 s. Such hyperthermia could, eliminate cancer cells efficiently within a short time.31


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Figure 2. (a) UV−vis−NIR absorption spectra of the Bi@PPy-PEG NHs dispersed in water (140 µg·mL−1) before and after placed at room temperature for a month. (b) Temperature (T) and (c) temperature variation (∆T) of the Bi@PPy-PEG NHs at different concentrations upon laser irradiation for 10 min. (d) Infrared thermal imaging of the Bi@PPy-PEG NHs suspensions during the laser irradiation.

Moreover, it was demonstrated that the strong photothermal effect of the Bi@PPy-PEG NHs can be also applied on infrared thermal (IRT) imaging (Figure 2d), where the high contrast and imaging intensity also relied on the concentration and irradiation time. The IRT imaging is capable to provide real-time monitoring of the PTT treatment.32 In addition, the photothermal conversion stability was examined from the NHs dispersion (100 µg·mL-1) upon five repeated irradiation cycles. For each cycle, the dispersion was irradiated for 180 s and then cooled with the laser off for 720 s.


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It was found that, the temperature rise was kept steady upon the repeated cycles (Figure 3a). Moreover, the optical absorption was also barely changed compared with that before the cycling (Figure 3b). All the results support the excellent photothermal stability of the NHs.

Figure 3. Photothermal stability of the Bi@PPy-PEG NHs. (a) Temperature of the Bi@PPy-PEG NHs (100 µg·mL-1) upon five repeated cycles of the NIR laser irradiation. (b) UV−vis−NIR absorption spectra of the Bi@PPy-PEG NHs before and after irradiation.

To quantitatively evaluate the photothermal conversion capability, we explored the photothermal conversion efficiency (η) of the Bi@PPy-PEG NHs. The Bi@PPy-PEG NHs suspension (100 µg·mL-1, 1.0 mL) was irradiated with the NIR laser for 20 min, and then the laser was switched off and the system was cooled naturally. The photothermal conversion efficiency was deduced from the heating and cooling temperature curves recorded by a thermocouple probe (Figure 4a). According to the standard method proposed previously in the literature,33 the photothermal conversion efficiency of the Bi@PPy-PEG NHs was calculated to ~ 46.3% (Figure 4b), superior to the reported Bi-based nanoagents, e.g. 18.3% for the Bi2S3-PEG nano-urchins and 31.1% for the Bi2Se3 spherical sponges.16,18 The high efficiency is likely to be attributed to the contribution of PPy, which is known for its excellent photothermal effect. We further explored the photothermal conversion capability of the bare Bi core for comparison, revealing a lower


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conversion efficiency of ~ 30.4% (Figure S8). Therefore, it can be concluded that, besides the enhanced physiological stability, proper surface engineering and coating of the Bi nanoparticles with the PPy-PEG layer can also largely improve the photothermal performance of the Bi@PPyPEG NHs, which may hold great potential for PTT application.

Figure 4. (a) Heating and cooling curves of the Bi@PPy-PEG NHs dispersed in water (100 µg·mL1

) and pure DI water. (b) Plot of cooling time as the function of negative natural logarithm of the

temperature driving force, where the time constant of the heat transfer is measured as τS=366.8 s.

3.3. In Vitro Cytotoxicity. As a PTA for anitumor treatments, high biocompatibility is crucial for clinical applications. We carefully examined the cytotoxicity of the Bi@PPy-PEG NHs on both normal cells (HUVEC cells) and cancer cells (4T1 cells). The cells were incubated with the Bi@PPy-PEG NHs suspensions at various concentrations (0, 10, 50, 100, 150, 200 µg·mL-1, 100 µL) for 24 h and 48 h, respectively, followed by a CCK-8 assay to examine the cell viability. It was turned out that, in all cases, the cell viability was above 90% (Figure 5a and 5b) after the 24 h or 48 h incubation with the Bi@PPy-PEG NHs, indicating no detectable cytotoxicity of the Bi@PPy-PEG NHs to normal cells or cancer cells. The behavior of hemolytic influence on RBCs with the Bi@PPy-PEG NHs at various concentrations (100 to 1000 µg·mL-1) was also investigated. After


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incubation with the NHs for 24 h, no detectable hemolysis of RBCs was detected even at a high concentration of 1000 µg·mL−1 (Figure S9), indicating the reliable blood biocompatibility.

Figure 5. Cell viability of (a) 4T1 cells and (b) HUVEC cells after incubation with the Bi@PPyPEG NHs dispersed in culture medium at various concentrations for 24 h and 48 h, respectively.

3.4. Photothermal Therapy Effect in Vitro. We then explored the in vitro IRT imaging and photothermal effect on 4T1 cells. As shown in Figure 6a, 4T1 cells were incubated with the Bi@PPy-PEG NHs dispersions (50 µg·mL-1, 1.0 mL) in the wells No. 1 and 3, while the well No. 2 only contained 4T1 cells. NIR laser irradiation was performed on wells No. 2 and 3. Certain distance was kept between the wells to reduce the thermal influence from the neighboring ones. As anticipated, before irradiation (0 min), there was no difference from the wells based on the IRT imaging. After irradiation for 3 min, only well No. 3 containing the NHs showed distinct IRT contrast, indicating the significant temperature rise.


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Figure 6. (a) Digital photo and infrared thermal images of a 96-well plate before and after 3 min laser irradiation. The wells No. 1 and 3 contain the Bi@PPy-PEG NHs (50 µg·mL-1, 100 µL) and 4T1 cells, well No. 2 contains 4T1 cells only. Only wells No. 2 and 3 are treated with laser irradiation. (b) Cell viability of 4T1 cells after incubation with the Bi@PPy-PEG NHs at various concentrations and irradiated by NIR laser for different duration.

The in vitro PTT efficacy of the Bi@PPy-PEG NHs was evaluated by measuring the cell viability of 4T1 cells after incubation with the NHs dispersions at different concentrations and irradiated by NIR laser for different duration. As shown in Figure 6b, the treatment combining the Bi@PPy-PEG NHs with NIR laser irradiation induced severe cell death, whereas either the Bi@PPy-PEG NHs or irradiation alone did not reduce the cell viability. For instance, at the concentration of 100 µg·mL-1, the Bi@PPy-PEG NHs can kill more than 90% of 4T1 cells upon 3min irradiation, revealing efficient therapeutic effect on cancer cells. Moreover, to intuitively demonstrate the PTT efficacy, fluorescence signal from cells staining with calcein AM (green) and PI (red) was recorded with an inverted fluorescence microscope to distinguish the living cells (green) and dead cells (red) after the different treatments. As shown in Figure 7, for the Bi@PPy-PEG NHs plus laser irradiation group, 4T1 cells in the irradiation spot were effectively destroyed while most cells outside the illumination zone retained alive. On the


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contrary, the samples of other groups exhibited vivid green fluorescence in almost the entire well without red signal, indicating that exposure of 4T1 cells to either NIR irradiation or the Bi@PPyPEG NHs alone did not compromise the cell viability. These results indicate that the Bi@PPy-PEG NHs can mediate the pronounced and spatial-selective photothermal destruction of cancer cells.

Figure 7. Fluorescence images of 4T1 cells treated by the Bi@PPy-PEG NHs only, NIR laser irradiation only, and the combination of the NHs and 5-min irradiation. Scale bar is 1000 µm.

3.5. In Vitro and In Vivo CT and PA Imaging. Due to the high X-ray attenuation coefficient of Bi element, the Bi@PPy-PEG NHs enriched Bi are anticipated to be a good CT contrast agent. We first studied the CT imaging performance of the Bi@PPy-PEG NHs by acquiring the CT phantom images of the NHs aqueous dispersions in vitro. It can be seen from the Figure 8a that the CT images became progressively brighter with the increased NHs concentrations, where the image brightness corresponds to the incremental CT signal intensity. Moreover, the CT value of each


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dispersion was further calculated to deduce the CT contrast-enhancement efficiency. Consistently, CT values of the Bi@PPy-PEG NHs increased linearly with the NHs concentration, resulting in an enhancement efficiency as high as ~ 14.4 HU·mM-1, which is 3.7 fold as that of iohexol (3.93 HU·mM-1), a commercial iodine-based CT contrast agent used in the clinic,34 and also higher than that of most other metal-based CT agents reported so far.35-37 To further explore the potential of the Bi@PPy-PEG NHs for in vivo CT imaging, Balb/c mice bearing tumors were intratumorally (i.t.) injected with the Bi@PPy-PEG NHs and then imaged by a small animal X-ray CT imaging system. The mice were scanned by CT scanning system before and after the injection. As expected, unlike the CT results before injection, the tumor site after injection of the NHs suspension showed strong CT response, indicating that the Bi@PPy-PEG NHs can perform as a high-performance CT agent in vivo. Given the high NIR absorbance and photothermal conversion capability, the possible application of the Bi@PPy-PEG NHs in PA imaging was also investigated. Similar to in vitro CT imaging results, more prominent brightness, i.e. higher PA signal intensity, was observed with the gradually-increased Bi@PPy-PEG NHs concentrations (Figure 8d and 8e), indicating the excellent PA imaging capability. For PA imaging experiments in vivo, compared with the signal of tumor site before the NHs injection, stronger PA signal with high contrast at the tumor site was obtained after the injection. We can therefore conclude that, besides the effective PTT therapeutic effect, the Bi@PPy-PEG NHs showed considerable potentials on multiple high-contrast imaging, including in vitro/vivo PA and CT imaging as well as IRT imaging.


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Figure 8. CT/PA imaging in vitro/vivo. (a) In vitro CT images and (b) linear fitting of the CT values of the Bi@PPy-PEG NHs at various concentrations as indicated. (c) In vivo CT images of mice before (pre) and after (post) injection with the Bi@PPy-PEG NHs. The tumor site is indicated by the white arrow. (d) In vitro PA images and (e) PA intensity of the Bi@PPy-PEG NHs at various concentrations as indicated. (f) In vivo PA images of mice before (pre) and after (post) injection with the Bi@PPy-PEG NHs. The tumor site is marked by the black cycle.

3.6. Biodistribution and PTT of the Bi@PPy-PEG NHs In Vivo. The distinct hypotoxicity and blood biocompatibility imply that the Bi@PPy-PEG NHs could be applied as a novel photothermal therapy agent. It has been addressed in the literature that nanoagents with a size in the range of 20200 nm are capable to accumulate at tumor sites through the enhanced permeability and retention


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(EPR) effect.38,39 The biodistribution of the NHs in tumor-bearing mice was detected by determining the Bi content with inductively coupled plasma-mass spectroscopy (ICP-MS), 24 h after intravenous (i.v.) injection of the Bi@PPy-PEG NHs (10.0 mg·mL-1, 200 µL). The result revealed a tumor uptake of Bi as high as ∼ 4.58 µg·g-1. Based on the tests of the major organs, the NHs were found to be mainly accumulated in the liver and spleen (Figure S10), which has been widely observed for other nanoagents.13,40 Furthermore, the PTT effect of the Bi@PPy-PEG NHs on tumor inhibition was performed in vivo. Tumor-bearing mice were divided into three groups, including (1) laser only, (2) NHs only, and (3) NHs + laser. 24 h after i.v. injection, the mice were irradiated by the NIR laser, and the temperature variation at different time points was recorded using an IR thermal camera (Figure 9a). After 15 min irradiation, the tumor temperature of ‘NHs + laser’ group rapidly reached ∼ 62.9 °C. Such a large temperature rise further confirms the high in vivo PTT efficacy and the efficient passive tumor accumulation of the NHs. After the treatments, the length and width of the tumors were measured every 2 days, and the tumor volumes were calculated and counted as a function of time (Figure 9b). The tumors for the ‘NHs + laser’ group were efficiently inhibited after the irradiation, while the tumors of the other two groups grew fast with time. 16 days later, the tumors were collected and weighted. The ‘NHs + laser’ group showed the lowest mean tumor weight (Figure 9c). These results demonstrated that the NHs can act as a competent photothermal agent for in vivo tumor inhibition. To explore the potential toxicity of the NHs-mediated PTT, all Balb/c mice were carefully monitored in the entire experimental period (such as eating, drinking, grooming, activity, and excretion), showing no abnormal signs. For all the groups, there is no evident difference on the growth trend of the mice body weight (Figure 9d). Moreover, 16 days after the PTT treatment, the


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major organs were collected and stained with H&E for histology analysis (Figure 9e). Comparing with the health mice, no noticeable inflammation or organic lesion was observed from the ‘NHs + laser’ group, confirming no/very low in vivo toxicity of the NHs at least in our tested doses. Furthermore, serum biochemistry assay was also carried out. The results showed that the parameters such as liver function markers (AST, ALP, ALT) and renal function markers (UA, CREA, BUN) were all within the normal ranges (Table S1), further suggesting the excellent biocompatibility of the Bi@PPy-PEG NHs.


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Figure 9. (a) Infrared thermal images of tumor-bearing mice recorded during laser irradiation. (b) Tumor growth curves and (c) average weight of tumors collected from the tumor-bearing mice 16 days after various treatment (n=3). (d) Body weight of mice after various treatments (n=3). Healthy mice were used as the control. (e) Histology analysis of the major organs 16 days after the PTT treatment.

4. CONCLUSIONS In summary, the core-shell Bi@PPy-PEG NHs have been successfully fabricated into a highperformance theranostic agent. It is demonstrated that the proper surface engineering of the bare Bi core by PPy-PEG shell coating results in not only a high physiological stability without detectable cytotoxicity but also a largely improved photothermal conversion capability for practical applications. The Bi@PPy-PEG NHs possess broad and high absorption in the NIR region and a photothermal conversion efficiency as high as ~ 46.3%, much superior to that of the bare Bi nanoparticles. Consequently, the NHs can induce a high-rate inhibition of cancer cells upon a short NIR irradiation in vitro/vivo. Thanking to the pronounced photothermal effect, the Bi@PPy-PEG NHs can perform as a high-contrast PA imaging agent both in vitro and in vivo. Meanwhile, these NHs enriched high-Z Bi element indeed show high performance on CT imaging, with a CT enhancement efficiency of 14.4 HU·mM-1, which is much higher than that of commercially available iodine-based and most other metal-based CT contrast agents reported previously. Such a biocompatible nanohybrid integrating high-performance PTT and multimodal imaging offers considerable promise for future clinical treatments to cancers.


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Supporting Information. Additional Figures including the absorption spectra of the bare Bi nanoparticles, chemical structure of DA-PEG, high-resolution TEM image of the Bi@PPy-PEG NHs, chemical stability of the Bi nanoparticles and Bi@PPy-PEG NHs in PBS with different pHs, photos of Bi@PPy-PEG NHs dispersed in various solutions, size distribution histograms of the Bi@PPy-PEG NHs dispersed in various physiological mediums for 1 or 9 days, absorption spectra of the Bi@PPy-PEG NHs in water and culture medium, the photothermal conversion efficiency of the Bi nanoparticles, hemolytic percent of RBCs incubated with Bi@PPy-PEG NHs at various concentrations for 24 h, in vivo biodistribution of the Bi@PPy-PEG NHs 24 h after i.v. injection, and table of serum biochemistry assay. The supporting information is available free of charge via the Internet at http://pubs.acs.org.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21473045, 51401066), the Fundamental Research Funds from the Central University (PIRSOF HIT A201503), and the State Key Laboratory of Urban Water Resource and Environment, the Harbin Institute of Technology (2018DX04).


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Multifunctional Bi@PPy-PEG Core-Shell Nanohybrids for Dual-Modal

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