Multifunctional Bismuth Selenide Nanocomposites for Antitumor

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Multifunctional Bismuth Selenide Nanocomposites for Anti-Tumor Thermo-Chemotherapy and Imaging Zhenglin Li, Ying Hu, Kenneth A. Howard, Tingting Jiang, Xuelei Fan, Zhaohua Miao, Ye Sun, Flemming Besenbacher, and Miao Yu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b06259 • Publication Date (Web): 14 Dec 2015 Downloaded from http://pubs.acs.org on December 14, 2015

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Multifunctional Bismuth Selenide Nanocomposites for Anti-Tumor Thermo-Chemotherapy and Imaging Zhenglin Li†, ‡, §, Ying Hu £, Kenneth A. Howard§, Tingting Jiang†, Xuelei Fan†, Zhaohua Miao¥, 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 150000, China ‡Condensed Matter Science and Technology Institute, Harbin Institute of Technology, Harbin 150000, China § Interdisciplinary Nanoscience Center (iNANO) and Department of Physics and Astronomy, Aarhus University, Aarhus 8000, Denmark £ School of Life Science and Technology, Harbin Institute of Technology, Harbin 150000, China ¥ School of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150000, China

E-mail: [email protected], [email protected], [email protected]

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Abstract To integrate real-time monitoring and therapeutic functions into a single nanoagent, we have designed and synthesized a drug-delivery platform based on a polydopamine(PDA)/human serum albumin (HSA)/doxorubicin (DOX) coated bismuth selenide (Bi2Se3) nanoparticle (NP). The resultant product exhibits high stability and biocompatibility both in vitro and in vivo. In addition to the excellent capability for both X-ray computed tomography (CT) and infrared thermal imaging, the NPs possess strong near-infrared (NIR) absorbance, and high capability and stability of photothermal conversion for efficient photothermal therapy (PTT) applications. Furthermore, a bimodal on-demand pH/ photothermal-sensitive drug release has been achieved, resulting in a significant chemotherapeutic effect. Most importantly, the tumor-growth inhibition ratio achieved from thermo-chemotherapy of the Bi2Se3@PDA/DOX/HSA NPs was 92.6%, in comparison to the chemotherapy (27.8%) or PTT (73.6%) alone, showing a superior synergistic therapeutic effect. In addition, there is no noticeable toxicity induced by the NPs in vivo. This multifunctional platform is, therefore, promising for effective, safe and precise anti-tumor treatment and may stimulate interest in further exploration of drug loading on Bi2Se3 and other competent PTT agents combined with in situ imaging for biomedical applications. Keywords: bismuth selenide nanoparticle, multifunctional nanocomposite, photothermal therapy, drug delivery, bioimaging.


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In the last decade, great efforts have been given to multifunctional nanoagents that combine diagnostic and therapeutic functions for high-efficient and low-toxicity anti-tumor treatments. In particular, combining real-time imaging with spatially precise photothermal therapy (PTT) mediated by inorganic nanoparticles responsive to near-infrared (NIR, λ=700-1100 nm) light through conversion of photo energy into heat, has attracted recent interest due to its simplicity, safety, non-invasiveness as well as targeting and remote-control properties.1-5 Furthermore, the imaging component allows identification of the location and size of tumor, detection of the accumulation of PTT agents and monitoring of the therapeutic effects.6-8 However, eradicating tumor tissues completely by PTT alone is difficult due to the light scattering and absorption in biological tissues.9-10 The combination of PTT with chemotherapy (termed thermo-chemotherapy) is, thus, attractive for enhanced and optimized anti-cancer efficacy.11-13 Integration of efficient drug loading and locational on-demand drug release together with high-contrast imaging on a competent PTT nanoagent, is crucial to improve the anti-tumor functions and avoid the requirements for multiple doses. Bismuth selenide (Bi2Se3) is one of the most typical topological insulators with a single Dirac cone and a relatively large bulk gap of 0.3 eV, showing great potential as the next generation of quantum computing, spintronic, optoelectronic devices.14 Although considerable attention has been paid in the physics, chemistry and materials fields, the application potentials of Bi2Se3 nanomaterials in the biomedical field have been ignored. Interestingly, Bi2Se3 is promising for biological applications supported by the use of bismuth (Bi) as a therapeutic agent15 and its high X-ray attenuation coefficient, whilst, selenium (Se) is a vital trace element and can reduce the occurrence and fatality of prostate, liver and lung cancers.16 Until very recently, the biocompatibility, metabolizability and the low toxicity of Bi2Se3 nanoplates were demonstrated in vivo.17 Most importantly, Li et al. unveiled the high potential of Bi2Se3 nanoplates for PTT and X-ray computed tomography (CT) imaging.18 A major challenge of the present Bi2Se3 nanoagents is their high oxidation and instability in vitro and in vivo, 17 which largely limit their practical applications. Moreover, the lack of appropriate surface functionalization and the


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difficulty of subsequent drug loading, have restricted the use of Bi2Se3 nanomaterials as drug vehicles as well. In this study, we have designed and fabricated a multifunctional nanoplatform applied simultaneously for PTT, chemotherapy and dual-modal imaging using human serum albumin (HSA) coated Bi2Se3 NP loaded with doxorubicin (DOX). As shown in Scheme 1, the Bi2Se3 core was produced in the presence of polyvinylpyrrolidone (PVP), sequentially sealed in a polydopamine (PDA) shell through in situ polymerization of dopamine (DA) by dispersing the as-grown cores in an alkaline DA solution and mildly shaking at room temperature to form Bi2Se3@PDA. PDA is a highly biocompatible and biodegradable polymer widely distributed in almost all living organisms and can spontaneously form a conformal layer on surfaces allowing further surface modification.19 Next, DOX was added along with Bi2Se3@PDA nanoparticles (NPs) into a HSA solution with sonication. In this way, the HSA layer can adsorb onto the aminated NPs surfaces through electrostatic forces,20 with DOX embedded and steadily bonded in the matrix of HSA by hydrogen bonding-based hydrophobic interaction.21-22 In this configuration, HSA rather than DOX is exposed to the biological environment before drug release. Multiple advantages of HSA coating on NPs have been reported, including reduced immunogenicity, prolonged circulatory half-life, low levels of mononuclear phagocyte system clearance and improved pharmacokinetic properties.20 Physicochemical properties including morphology, size distribution, chemical composition, and stability of Bi2Se3@PDA/DOX/HSA NPs were determined. Furthermore, light absorption, X-ray attenuation capability, NIR imaging, photothermal energy conversion, drug loading, and drug release profiles were investigated. Cellular uptake and internalization, effects of chemotherapy, PTT and thermo-chemotherapy, and toxicity in vitro and in vivo were explored to evaluate the biological effects of the nanocomposites. We demonstrate that the coating successfully protected the NPs from oxidation and the NPs were highly stable and well dispersed in diverse physical solutions over a long period. In addition to the high X-ray attenuation capability for CT imaging, the strong absorbance in NIR region, excellent capability and stability of


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photothermal conversion support the application of the Bi2Se3@PDA/DOX/HSA NPs for PPT treatment and infrared thermal imaging. Furthermore, a bimodal pH- and photothermal-sensitive drug release in a controllable fashion has been realized, showing significant tumor inhibition. Moreover, the synergistic effect of thermo-chemotherapy was substantially superior over either mono-treatment. The NPs showed low toxicity, without evident damage or inflammatory lesion in all major organs. This potent multifunctional platform is, therefore, very promising and favorable for effective, precise and safe antitumor treatment in clinic. And such facile drug-loading method may be used for incorporation of other PTT agents into alternative drug delivery vehicles.

Scheme 1. Schematic illustration of the synthesis and multifunction of Bi2Se3@PDA/DOX/HSA NPs.

RESULTS AND DISCUSSION Characterization of Bi2Se3@PDA/DOX/HSA NPs. The morphology of as-grown Bi2Se3, Bi2Se3@ PDA and Bi2Se3@PDA/DOX/HSA NPs was determined by transmission electron microscopy (TEM, Figure 1 and Figure S1), atomic force microscopy (AFM, Figure 1) and scanning electron microscopy (SEM, Figure S2). As shown in Figure 1A, the Bi2Se3 cores appeared circular with varied surface thickness. Three-dimensional AFM (Figure 1D) further reveals that the core actually formed a nanoplate-like shape composed of two layers, i.e. a circular lower layer and a flat-top upper layer. The ACS Paragon Plus Environment

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high-resolution TEM image in Figure 1B shows a hexagonal lattice fringe with a spacing of 0.21 nm, corresponding to that of the Bi2Se3 (110) plane.23 The selected area electron diffraction (SAED) pattern in Figure 1C indicates that the growth is along [001] zone axis. The crystallization nature was examined by powder X-ray diffraction (XRD, Figure S2C), and all peaks in the pattern can be assigned to the reflections of Bi2Se3 rhombohedral phase (JCPDS Card No. 33-0214).18, 23 In addition, energy dispersive spectroscopy (EDS) analysis (Figure S2D) confirms the presence of Se and Bi elements.

Figure 1. (A) TEM, (B) high-resolution TEM, (C) SAED pattern and (D) three-dimensional AFM results of as-grown Bi2Se3 cores. (E-G) Histograms of the lateral size of Bi2Se3, Bi2Se3@PDA and Bi2Se3@PDA/DOX/HSA NPs based on TEM results. (H-J) AFM images and line scans of Bi2Se3, Bi2Se3@PDA and Bi2Se3@PDA/ DOX/HSA NPs.

After coating, the Bi2Se3@PDA and Bi2Se3@PDA/DOX/HSA NPs appeared similar, with a polymer shell fully-wrapping around the Bi2Se3 core (Figure S1). Based on the statistical results from the TEM images, the particle size was increased following coating, from ~ 84 nm for Bi2Se3 to ~ 104


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nm for Bi2Se3@PDA and ~ 112 nm for Bi2Se3@PDA/DOX/HSA NPs (Figure 1E-G). AFM results show the average thickness was raised from ~ 14.2 nm for Bi2Se3 to ~ 22.8 nm for Bi2Se3@PDA and ~ 25.3 nm for Bi2Se3@PDA/DOX/HSA accordingly (Figure 1H-J). The coated NPs exhibited high uniformity, telling from the low polydispersity index (PDI) of Bi2Se3@PDA (0.109) and Bi2Se3@ PDA/DOX/HSA (0.107) measured by dynamic light scattering (DLS). In addition, the zeta potential (ζ) varied with the coating as well, showing a negative charge of approximately –15.3 mV for the naked Bi2Se3, a positive potential of +3.7 mV for Bi2Se3@PDA NPs and a negative charge of –14.4 mV when further adsorbed with negatively charged HSA. As illustrated in Ultraviolet-visible-near infrared (UV-vis-NIR) absorption spectra (Figure 2A), pronounced adsorption in the range of 200-900 nm was observed for both Bi2Se3 and Bi2Se3@PDA/DOX/HSA NPs, with the latter higher due to the presence of PDA layer. The loading of DOX was further confirmed by both fluorescence emission spectra (Figure 2B) and Fourier transforminfrared spectroscopy (FT-IR, Figure S4A). Given that Bi2Se3, PDA and HSA have no fluorescence emission, the distinct emission peak of Bi2Se3@PDA/DOX/HSA NPs at 587 nm together with the two shoulders is most probably attributed to DOX. And the emission intensity was increased with the NPs concentration. Using the corresponding standard calibration curve, the saturated loading efficacy was calculated to be ~ 5.2 % (Figure S6).

Figure 2. (A) UV-vis-NIR absorption spectra of Bi2Se3 and Bi2Se3@PDA/DOX/HSA NPs. (B) Fluorescence emission spectra of Bi2Se3 and Bi2Se3@PDA/DOX/HSA NPs suspension with excitation at 480 nm.


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In addition, the Bi2Se3@PDA/DOX/HSA NPs exhibited excellent stability and good dispersion in water, phosphate buffer saline (PBS) and Dulbecco’s modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), without any macroscopic aggregates (Figure S4B), which was supported by the perfect linear increase of absorbance with the NPs concentration (Figure S4C and S4D). Similar to previously reports,17 the naked Bi2Se3 NPs in this work were also unstable, showing sediment at the bottom of the sample bottle and color change from black to brown a few days after dispersion in water and kept at room temperature. On the contrary, the color and dispersion of Bi2Se3@PDA/DOX/HSA NPs remained unchanged over 6 weeks. This was further confirmed by the UV-vis-NIR absorption spectra (Figure S5). While the adsorption over a wide range of 300-900 nm for Bi2Se3 was largely reduced compared with that for the fresh sample, the absorption profile of Bi2Se3@PDA/DOX/HSA NPs was barely changed over 6 weeks, revealing the much improved stability and anti-oxidative property of the coated NPs hence their much improved competency for practical applications. Photothermal properties of the Bi2Se3@PDA/DOX/HSA NPs. The photothermal conversion capability of Bi2Se3@PDA/DOX/HSA NPs was investigated by monitoring the temperature rise under the irradiation of 808 nm NIR laser at 1.2 W/cm2 using a thermocouple probe. As depicted in Figure 3A, for DI water and aqueous solutions of DA, HSA and DOX, temperature was barely changed (less than 1.5°C) upon irradiation. In contrast, the temperature of Bi2Se3 and Bi2Se3@PDA/DOX/HSA suspensions (100 µg/mL) was strikingly increased to 47.4°C and 50.0°C over a 10-min period (Figure 3A). As shown in Figure 3B and 3C, the temperature rise followed a time- and concentration-dependent manner. In particular, at a NP concentration of 200 µg/mL and under irradiation less than 4 min, the temperature can reach 43.0°C, the critical temperature required to induce the death of cancer cells.24, 25 Given the body temperature is normally already 36-37°C, heating up to 43.0°C or higher is, therefore, easy to achieve even at a much lower concentration or a shorter irradiation period. Besides the conversion capability, the conversion stability is another vital factor to evaluate photothermal agents. For instance, some Au-related photothermal materials show a serious decline in the maximum


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temperature over irradiation cycles.26 In the case of Bi2Se3@PDA/DOX/HSA NPs, the temperature elevation (Figure 3D) and particle morphology (Figure S7) were perfectly maintained upon multiple irradiation cycles, which is likely due to the strong chemical bonds in Bi2Se3 and recurrence of charge transfer. The remarkable efficiency and stability on photothermal conversion support the Bi2Se3@PDA/DOX/HSA as an excellent candidate for PTT applications.

Figure 3. Photothermal properties of Bi2Se3@PDA/DOX/HSA NPs. (A) Temperature elevation of different aqueous solutions (as indicated) upon irradiation. (B) Temperature elevation of Bi2Se3@PDA/DOX/HSA NPs suspensions at various concentrations as a function of irradiation time. (C) Plot of temperature variation upon a 10-min irradiation versus the concentration of the Bi2Se3@PDA/DOX/HSA NPs. The inset: infrared thermal images of 100 µg/mL Bi2Se3@PDA/DOX/HSA suspension irradiated for 0 min (left) and 10 min (right). (D) Temperature elevation of Bi2Se3@PDA/DOX/HSA NPs (100 µg/mL) over 5 irradiation cycles.

Infrared thermal imaging and CT imaging. Following determination of the excellent photothermal properties, the infrared thermal imaging capability of Bi2Se3@PDA/DOX/HSA NPs was then


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investigated. As illustrated in Figure 4A-4B, Hela cells were incubated with Bi2Se3@PDA/DOX/HSA NPs (Wells No.1, 2, 5, and 6) and pure DMEM (Wells No. 3 and 4) respectively. Wells No. 3-6, marked by a circle, were irradiated by the 808-nm laser. As anticipated, only the wells containing the NPs under irradiation (No.5-6) provided bright thermal image, indicating that Bi2Se3@PDA/DOX/HSA NPs can act as excellent contrast agents for infrared thermal imaging and realize real-time monitoring of thermal dynamics in a PTT process. Given the high X-ray attenuation coefficient of Bi element (Bi: 5.74 cm2kg-1 vs the CT contrast agent used in clinic, I: 1.94 cm2kg-1 at 100 keV), the feasibility of the NPs for CT imaging was evaluated by phantom images taken at different NPs concentrations in vitro. The incremental CT signal intensity became progressively stronger with the NPs concentrations (Bi concentrations: from 0 to 45.6 mM) (Figure 4). CT value (Hounsfield Unit, HU) at each concentration was calculated and summarized in Figure 4D, showing a monotonic linear-increase with the concentration. The X-ray absorption coefficient of the Bi2Se3@PDA/DOX/HSA NPs was calculated to be 7.53 HU mmol/L, which is significantly higher than that of the commercial contrast agent (iohexol, 3.93 HU mmol/L).27 The enhanced CT contrast efficacy of Bi2Se3@PDA/DOX/HSA NPs is promising to reduce the potential side-effects and complications caused by the high dose of contrast agents in clinical applications. Motivated by the high X-ray attenuation capability of the Bi2Se3@PDA/DOX/HSA NPs in vitro, we then studied their performance as the CT contrast agent in vivo. Anesthetized tumor-bearing Balb/c mice were intratumorally (i. t.) injected with the Bi2Se3@PDA/DOX/HSA NPs (5.0 mg/mL, 100 µL) and imaged using an animal micro-CT system. As demonstrated by the three-dimensional (3D) CT images in Figure 4E, the imaging contrast can be largely enhanced by the injection, showing a much higher mean CT value in the tumor site. In addition, the CT images taken from tumor-bearing Balb/c mice treated by intravenous (i. v.) injection of Bi2Se3@PDA/DOX/HSA NPs (5.0 mg/mL, 200 µL) at indicated time points were presented in Figure S8. Due to the passive accumulation of the NPs into tumor through the enhanced permeation and retention (EPR) effect over time, the mean HU value can


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be increased gradually from ∼54.0 HU before injection to ∼72.3 HU at 1 h, ∼92.4 HU at 5 h, ∼126.8 HU at 18 h, and ∼118.3 HU at 24 h after i. v. injection. Moreover, largely enhanced CT contrast signals in the liver and spleen of mice were also noted after i.v. injection, suggesting the uptake of the NPs by the mononuclear phagocyte system. It can be, therefore, concluded that Bi2Se3@PDA/DOX/HSA NPs may be a promising agent for CT imaging in vivo.

Figure 4. Infrared thermal imaging of a 96-well cell-culture plate containing HeLa cells and Bi2Se3@PDA/DOX/HSA (Well No. 1, 2, 5, and 6) or Hela cells only (No. 3 and 4) (A) before and (B) after laser irradiation for 5 min, where the irradiated region is marked by the circle. (C) In vitro CT contrast images of Bi2Se3@PDA/DOX/HSA at different NPs concentrations. (Bi concentration: 0, 4.56, 13.7, 27.4, 36.5, 45.6 mmol/L, respectively). (D) Linear fitting of the CT value as a function of Bi concentration (R2 = 0.993). Three-dimensional in vivo CT images of mice (E) before (Pre) and (F) after (Post) intratumoral injection of Bi2Se3@PDA/DOX/ HSA NPs (5.0 mg/mL, 100 µL). The CT contrast of the section corresponding to the tumor is largely enhanced after the injection.


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pH- and photothermal-sensitive drug release. The capability of drug release of Bi2Se3@PDA/DOX/ HSA NPs was first examined in PBS buffers at various pH (pH= 4.5, 5.5, 7.4). As shown in Figure 5A, drug release was pH-dependent and increased in acidic environments. This may be attributed to the increased protonation of the amino group in DOX under acidic conditions.22, 28 The release of free DOX molecules was examined as a control group, showing a complete cumulative release within 14 h. In contrast, the drug release from Bi2Se3@PDA/DOX/HSA NPs was slower and can be sustained over 190 h. Since the microenvironment at tumor sites in vivo and inside endosome/lysosome of cells is acid (pH= 4.5-6.0) while the bloodstream is neutral,29 the preferential DOX release at acid environments can minimize the unwanted risk to normal cells during the intravenously injected NPs are circulated in vivo. When the NPs are accumulated at tumor sites by the EPR effect and processed in endosome and lysosome after penetrated the cells, the low pH may benefit the on-demand chemotherapy.

Figure 5. (A) DOX release from Bi2Se3@PDA/DOX/HSA NPs at different pH as a function of cumulative time. The release profile of free DOX molecules at pH 7.4 is given as comparison. (B) Temperature-triggered release of DOX from Bi2Se3@PDA/DOX/HSA NPs. The samples at various pH were irradiated with the NIR laser for 8 min at different time points indicated by the arrows. Error bars are based on at least triplicate measurements.

To investigate the photothermal influence on drug release, Bi2Se3@PDA/DOX/HSA dispersions at pH 4.5, 5.5 and 7.4 respectively were exposed to the NIR laser for 8 min. DOX released, before and after the irradiation, was collected and measured. As depicted in Figure 5B, in all cases the release rate can be significantly varied in a controllable manner by switching the laser on or off. The release was


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potentiated in the acidic environment, and evident thermo-triggered burst of DOX occurred at pH = 4.5 and 5.5 even over multiple cycles of irradiation. In contrast, only limited DOX was released at pH = 7.4. Cellular

uptake

and

internalization.

The

cellular

uptake

and

internalization

of

Bi2Se3@PDA/DOX/HSA NPs for the intracelluar delivery of DOX were investigated by addition to either human umbilical vein endothelial cells (HUVEC, a normal cell line) or HeLa cells (the most commonly used human cell line derived from cervical cancer). 50 µg/mL Bi2Se3@PDA/DOX/HSA NPs dispersions or free DOX molecules at an equivalent concentration were added for 2 h or 4 h, and irradiated by the NIR laser for 5 min. After the treatment, the cells were stained using 4',6-diamidino-2phenylindole (DAPI) and immediately imaged using a fluorescence microscope. In the obtained images, the blue and red fluorescence signals are attributed to the DAPI-stained nuclei and DOX, respectively. As shown in Figure S9A, after incubated with free DOX molecules, domains covered by DOX-signal perfectly matched the DAPI-stained section, indicating that free DOX predominantly targeted the nucleus. In contrast, the red fluorescence was detected within both the nucleus and the cytoplasm (Figure 6) when incubated with Bi2Se3@PDA/DOX/HSA NPs as early as 2 h. It was reported previously that NPs can be taken up by cell endocytosis and subsequently delivered to lysosome in cytoplasm.30 The red signal from cytoplasm, therefore, may correspond to the NPs attached DOX, and the aggregated dot-like DOX signals in the cytoplasm then indicate the efficient cellular uptake of the Bi2Se3@PDA/DOX/HSA NPs. The acidic environment in lysosome may facilitate effective pHsensitive drug release of the NPs, which was confirmed by the intense red fluorescence of released DOX at nucleus. It is noted that the DOX signal in HUVEC cells was much weaker than that in HeLa cells, which is relevant to the specific alteration of cancer cells relative to healthy cells, such as improved permeability of cell membrane due to less glycoproteins, higher metabolic rate and increased expression of lysosomal enzymes.31-34 In addition, the cellular uptake of Bi2Se3@PDA/DOX/HSA NPs was time- and thermo-dependent, that was much more significant when the incubation time was extended from 2 h (Figure 6B) to 4 h


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(Figure 6C) or the temperature was increased from 4 °C to 37 °C (Figure 6C-D). Considerably enhanced intracellular DOX fluorescence in both cytoplasm and nucleus was visual in Bi2Se3@PDA/DOX/HSA NPs-incubated HeLa cells after 5-min NIR irradiation (Figure 6E) in comparison with those without irradiation (Figure 6B). In fact, the hyperthermia provided by the NPs can not only increase the cellular internalization of the NPs through promoted cellular metabolism and membrane permeability,35 induce protein denaturization25 and partial or even full disruption of lysosomal membrane to release the lysosome enzymes,36 but also trigger more release of free DOX from the internalized NPs inside the cells. In contrast, for the control group treated with free DOX, no significant change took place in the cells after irradiation (Figure S9B).

Figure 6. Fluorescence images of (A) HUVEC and (B-E) HeLa cells treated with Bi2Se3@PDA/DOX/HSA NPs with/without laser irradiation for various durations. Except (D), all other results were collected at 37°C. Scale bar is 100 µm.


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The chemotherapy effect in vitro. To examine the inhibitory effect on cancer cells, bare Bi2Se3 and Bi2Se3@PDA/DOX/HSA NPs suspensions were added to HeLa cells for 2 h, 12 h, and 24 h, using various NPs concentrations and imaged by an optical microscope (Figure S10C-D). Compared with untreated cells (Figure S10A), cells exposed to Bi2Se3 nanoplates remained unchanged (Figure S10B) due to their excellent biocompatibility of Bi2Se3. In comparison, when incubated with Bi2Se3@PDA/DOX/HSA NPs, the amount of living cells was markedly decreased with incubation time, which is attributed to the cytotoxic effect of DOX released from the NPs. A Cell Counting Kit-8 (CCK-8) assay was used to quantify the viability of HeLa cells incubated with free DOX, Bi2Se3, Bi2Se3@PDA/HSA or Bi2Se3@PDA/DOX/HSA for different durations. Over a wide concentration range from 0 to 200 µg/mL, ∼ 90% of HeLa cells remained viable, even after a 48-h incubation with the Bi2Se3 and Bi2Se3@PDA/HSA (Figure 7A-B and S11), confirming negligible cytotoxicity. Moreover, based on the results for NPs concentration > 100 µg/mL, it suggests that the coating of PDA/HSA can further lower the cytotoxicity of Bi2Se3. In contrast, the cell viability was sharply decreased after incubation with Bi2Se3@PDA/DOX/HSA NPs, in a concentration- and timedependent manner. For example, at a concentration of 100 µg/mL, more than 88% of Hela cells were killed after 24-h incubation; at a concentration of as low as 20 µg/mL, only around 22% of the cells survived after 48-h incubation. Notably, compared with free DOX, the inhibition effect of Bi2Se3@PDA/DOX/HSA NPs at an equivalent concentration appear more effective (Figure 7C and S11), seemingly benefiting from the efficient uptake of the NPs by the cells together with the sensitive drug release of the NPs. Since DOX is able to induce apoptosis by non-specific oxidative damage to both outer and inner mitochondrial membranes as well as inhibition of topoisomerase II,37 the cytotoxic effect induced by Bi2Se3@PDA/DOX/HSA NPs NPs was further investigated using an apoptosis assay. HeLa cells were incubated with Bi2Se3, free DOX, or Bi2Se3@PDA/DOX/HSA NPs, respectively, stained with DAPI and then visualized by fluorescence microscopy. Compared with the cells treated by Bi2Se3, where no


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apparent change was observed on nuclei morphology (Figure 7D), cells incubated with either Bi2Se3@PDA/DOX/HSA NPs (Figure 7E-7H) or free DOX (Figure 7I) showed seemingly karyopyknosis with evident abnormal fragments (pointed out by yellow arrows), which are characteristic features of apoptosis.37, 38 The phenomenon became more pronounced at larger doses. These results suggest that DOX delivered by Bi2Se3@PDA/DOX/HSA can effectively destroy cancer cells by inducing apoptosis.

Figure 7. Cell viability of HeLa cells after incubation with Bi2Se3, Bi2Se3@PDA/HSA and Bi2Se3@PDA/DOX/HSA at various concentrations for (A) 24 h and (B) 48 h. (C) Cell viability of HeLa cells after incubation with Bi2Se3@ PDA/DOX/HSA NPs and free DOX at different concentrations for 24 h or 48 h. Image of HeLa cells incubated with (D) 20 µg/mL Bi2Se3; and Bi2Se3@PDA/DOX/HSA NPs at a concentration of (E) 5 µg/mL, (F) 10 µg/mL, (G) 15


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µg/mL and (H) 20 µg/mL; (I) Free DOX. Scale bar is 200 µm. Abnormal nuclear morphology is indicated by the yellow arrows.

PTT effect in vitro. To study the localized photothermal effect in vitro, HeLa cells were exposed to the laser for 0, 5 or 10 min immediately after addition of Bi2Se3@PDA/DOX/HSA NPs dispersions (1.0 mL per well, 50.0 µg/mL) or in absence of the NPs, followed by staining with calcein acetoxymethyl ester (Calcein AM) and propidium iodide (PI) to visualize viable and dead cells, respectively, using an inverted fluorescence microscope. Samples treated by either irradiation only or NPs alone (Figure 8A and 8B) exhibited vivid green fluorescence in the entire well without red signal. In contrast, the combination of Bi2Se3@PDA/DOX/ HSA NPs and irradiation induced high cell death (Figure 8C and 8D). After only 5-min irradiation, cells within the irradiation spot were effectively destroyed while most cells outside retained viable. When the irradiation was extended to 10 min, cancer cells were completely destroyed due to heat conduction.

Figure 8. Photothermal destruction of HeLa cells treated with (A) laser only, (B) Bi2Se3@PDA/DOX/HSA NPs only and (C, D) combination of Bi2Se3@PDA/DOX/HSA NPs and irradiation for 5 and 10 min respectively. Scale bar is 1000 µm. (E) Cell viability after treatment at various concentrations of Bi2Se3@PDA/DOX/HSA NPs upon irradiation


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for different duration. (F) The synergistic therapeutic efficacy under external stimulus by NIR irradiation.

We further evaluated the photothermal cytotoxicity of Bi2Se3@PDA/DOX/HSA NPs on HeLa cells using CCK-8 assay (Refer to Figure 8E). Before evaluation, a control experiment was performed to rule out the possible disturbance from the thermo-senstive drug release of the NPs. Hela cells were treated using free DOX at a concentration of 1.0 µg/mL (around 10 times as the DOX concentration released from 40 µg/mL NPs upon a 10-min irradiation), followed by a NIR irradiation for 10 min, and then washed with PBS for cell viability assay immediately. It was revealed that more than 91.6±2.7 % Hela cells remained alive. This indicates directly that, the cell viability can barely be affected by the thermosensitive drug release from the NPs during such a time scale. As seen from Figure 8E, compared with the untreated group, the viability of HeLa cells was only reduced by 6.7% after incubated with 40 µg/mL Bi2Se3@PDA/DOX/HSA NPs solution for 20 min. In contrast, when simultaneously treated with Bi2Se3@PDA/DOX/HSA NPs and the irradiation, the cell viability was decreased rapidly upon an irradiation as short as 5 min as long as the NP concentration was higher than 5 µg/mL. For instance, at a concentration as low as 20 µg/mL, the cell viability of HeLa cells under 5- and 10-min irradiation was only 25.7±2.2% and 8.3±1.8%, respectively (Figure 8E). These results in total suggest that Bi2Se3@PDA/DOX/HSA NPs have a significant photothermal therapeutic effect and may act as an effective agent for PTT applications. Thermo-chemotherapy in vitro and in vivo. To further demonstrate the synergistic therapeutic efficacy in vitro under external stimulus by NIR irradiation, the cell viability of HeLa cells were determined after incubated with Bi2Se3@PDA/HSA, Bi2Se3@PDA/DOX/HSA NPs and free DOX (Bi2Se3@PDA/DOX/HSA NPs and free DOX were normalized to be the equivalent DOX concentration) for 12 h and then under NIR irradiation. As shown in Figure 8F, the combination of Bi2Se3@PDA/DOX/HSA NPs and laser irradiation had a much higher potency to destroy HeLa cells at all the tested concentrations, than either photothermal therapy (Bi2Se3@PDA/HSA NPs) or the


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chemotherapy (free DOX), e.g. inducing an inhibition rate as high as 230.3% and 226.1% of the Bi2Se3@PDA/HSA and free DOX groups’ results at an equivalent concentration of 20 µg/mL. The largely improved therapeutic effect can be attributed to the increased cell uptake and drug release at elevated temperature, as revealed by the results in Figure 6. In order to explore the blood circulation and biodistribution of Bi2Se3@PDA/DOX/HSA NPs in vivo after i.v. injection to HeLa tumor-bearing Balb/C mice, blood samples and major organs of mice were severally collected, and solubilized by HNO3/H2O2 solution (HNO3:H2O2 = 2:1) for ICP-OES measurement of Bi element at selected time points. As shown in Figure S12A, the NPs exhibited a relatively long blood circulation with a half-life of 3.8 h, which is nearly three times as that of bare Bi2Se3 reported previously.17 Even 12 h and 24 h after the injection, the level of Bi3+ in blood was kept as high as 8.15% and 6.81% ID/g, respectively. Moreover, high levels of Bi element were detected in the tumor, as well as in the liver and spleen. The tumor uptake reached the peak value of 6.20% ID/g at 12 h post injection (Figure S12B). Such prolonged circulation half-life and preferential tumor accumulation are attributed to the HSA coating of the NPs.21-22 Encouraged by the synergistic anti-tumor effect in vitro, we then carried out in vivo studies to investigate the inhibiting tumor effectiveness of Bi2Se3@PDA/DOX/HSA NPs. HeLa cells (1×106) suspended in 100 µL PBS were subcutaneously injected into the back of each Balb/C female nude mouse (3 mice per group). Tumors with the mean diameter of approximately 8-10 mm were used in this study. The mice were divided into five groups as follows: (1) saline + NIR as the control group; (2) free DOX + NIR group; (3) Bi2Se3 + NIR group; (4) Bi2Se3@PDA/DOX/HSA only; (5) Bi2Se3@PDA/DOX/HSA + NIR group. 12 h after intravenous (i.v.) injection with saline, free DOX, Bi2Se3, or Bi2Se3@PDA/DOX/HSA NPs (~ 0.81 mg/kg in terms of DOX, and ~ 25 mg/kg in terms of Bi2Se3), the tumors were irradiated for 10 min (0.64 W/cm2).


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Figure 9. Comparative investigation of inhibiting tumor effectiveness in vivo. Group 1: saline + NIR laser; Group 2: free DOX + NIR laser; Group 3: Bi2Se3 + NIR laser; Group 4: Bi2Se3@PDA/DOX/HSA NPs only; Group 5: Bi2Se3@PDA/DOX/HSA NPs + NIR laser. (A) Infrared thermal images of HeLa tumor-bearing mice i.v. injected with saline, free DOX, Bi2Se3 nanoplates, and Bi2Se3@PDA/DOX/HSA NPs under NIR laser radiation. (B) Temperature variation of tumors monitored by the IR thermal camera on different groups during laser irradiation. (C) Growth curves of tumors after various treatments. The relative tumor volumes are normalized to their initial sizes. (D) Relative tumor growth ratio, (E) tumor growth inhibition ratio, and (F) body weight of mice from different groups.

The infrared thermal imaging and temperature variations in vivo at different time points during irradiation were monitored by an IR thermal camera. As shown by the colour bar in Figure 9A, the temperature raised from 20°C to 50°C is represented by purple, indigo, blue, green, yellow, orange, to red and white. It is found that before irradiation (i.e. at 0 s), all tumors showed a green color. Upon 10min irradiation, the color of saline or free DOX group was barely changed. In contrast, for the groups


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injected with Bi2Se3 or Bi2Se3@PDA/DOX/HSA, the tumor site turned red and became brighter upon extended irradiation, consistent with the temperature variation as shown in Figure 9B. The tumor temperature varied very little for the group of saline or free DOX (∆TSaline = 4.2 °C, ∆TFree DOX = 4.7 °C); whilst for Bi2Se3 or Bi2Se3@PDA/DOX/HSA group, upon a 10- min irradiation the temperature was rapidly increased to 47.0-49.0°C, which is sufficient to induce hyperthermia as well as triggered drug release based on the in vitro studies presented above. It can, thus, be concluded that Bi2Se3@PDA/ DOX/HSA NPs can provide effective PTT function and high-contrast real-time infrared thermal imaging in vivo. Following the treatments, the length and width of the tumors were measured by a digital caliper every 2 days for a period of 12 days and the relative tumor volumes (V/V0) were then plotted as a function of time. As shown in Figure 9C and Figure 10, the tumors i.v. injected with either saline or free DOX grew rapidly, which is consistent with previous findings that free DOX at such a low dose is not effective in inhibiting the tumor growth.39 The growth of tumors could be slightly reduced by Bi2Se3@ PDA/DOX/HSA NPs alone without laser irradiation, resulting in a smaller V/V0 (26.8) compared with that of the control group treated with saline (36.1). Pronounced tumor inhibition effect was already obtained for the PTT of Bi2Se3 alone, where the V/V0 (13.3) is only 36.9% as that of the control group. Importantly, more inhibition was achieved from the group of Bi2Se3@PDA/DOX/HSA + NIR, with a V/V0 value as low as 3.4, which was only 9.3% as that of the control group. Importantly, it is noted that although the tumor growth can be effectively inhibited by the PTT effect of Bi2Se3 alone in the first 6 days, a rapid recover of tumor growth was evident afterwards; whilst, the Bi2Se3@PDA/DOX/ HSA+NIR group maintained more than 90% inhibition ratio within the entire experimental period (16 days since treatments). As shown in Figure 9D-E, the tumor-growth inhibition ratio of Group 5 was 92.6%, while that for chemotherapy and PTT alone was only 27.8% and 73.6%, respectively. These in vivo results directly demonstrate that the inhibition efficacy of the thermo-chemotherapy produced by Bi2Se3@ PDA/DOX/HSA NPs is considerably higher than either monotherapy alone.


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Figure 10. Representative photos of mice before treatment and 10 days after various treatments. Group 1: saline + NIR as a control group; Group 2: free DOX+NIR; Group 3: Bi2Se3+NIR; Group 4: Bi2Se3@PDA/DOX/HSA only; Group 5: Bi2Se3@PDA/DOX/HSA + NIR.

Toxicity in vivo. Since the potential toxicity of the nanoagents in vivo is a crucial issue for practical applications, the behaviors of Balb/c nude mice (3 mice per group) after i.v. injection and laser irradiation were carefully monitored. No obvious signs of toxic effects, such as eating, drinking, grooming, activity, exploratory behavior, urination, or neurological status took place within 16 days. Moreover, the body weight of the mice was measured during the experimental period, showing no obvious variation upon different treatments (Figure 9F). The maintained body weight also indicates that there is no noticeable systemic toxicity of the combined therapy in vivo. In addition, since considerable localization of the NPs in the mononuclear phagocyte systems such as liver and spleen was revealed by both the in vivo CT imaging (Figure S8) and biodistribution results (Figure S12), histology analysis of the major organs such as heart, liver, spleen, lung and kidney stained with hematoxylin and eosin (H&E) were performed 16 days after treatments to evaluate the potential toxicity effects on these major organs. As shown in Figure 11, there was no evident organ damage or inflammatory lesion in all major organs, further confirming no/very low toxicity of the NPs in vivo at our tested dose. To further assess the potential toxicity in vivo, three groups of mice, including untreated healthy group and tumor-bearing mice treated by PBS and thermo-chemotherapy of Bi2Se3@PDA/DOX/HSA NPs, were sacrificed for serum biochemistry assay 16 days after the treatment. The results are


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summarized in Table S1. Despite the accumulation of NPs in the liver (Figures S8 and S12), the rather similar values of hepatic function markers (ALT, ALP, AST) between the thermo-chemotherapy group and the healthy mice indicate that the liver function was not influenced by the NPs within the experimental period. Furthermore, renal function markers (CREA, UA, and BUN) and other measured biochemical parameters also fit in normal ranges, further confirming no noticeable renal dysfunction or other side effects induced by the thermo-chemotherapy of Bi2Se3@PDA/DOX/HSA NPs. In addition, routine blood examination from the three groups of mice showed no increase of white blood cells (WBC) after the thermo-chemotherapy treatment compared with the group treated by PBS only, while all other indexes including red blood cells (RBC), hematocrit (HCT), hemoglobin (Hgb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelets (PLT) were similar relative to those of healthy mice, showing no detectable inflammatory response either. Although more studies are still required to systematically examine the potential long-term toxicity of Bi2Se3@PDA/DOX/HSA NPs, our preliminary data collectively provide evidence that Bi2Se3@PDA/DOX/HSA NPs at the given dose induce no noticeable toxicity in vivo and may hold promising potentials for clinic medical applications.

Figure 11. Histology analysis of the major organs of the mice 16 days after various treatments.


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Conclusion We have successfully designed a drug-delivery nanoagent based on PDA/HSA/DOX coated Bi2Se3, capable of simultaneous thermo-chemotherapy and real-time imaging. The resultant NPs exhibit high stability, safety, and biocompatibility both in vitro and in vivo. In addition to the capability of highcontrast CT and infrared thermal imaging, the Bi2Se3@PDA/DOX/HSA NPs possess photothermal properties, bimodal sensitive drug release, and excellent chemotherapy and PTT effects for tumor inhibition and eradication. The thermo-chemotherapy provided by the NPs is considerably more effective than PTT or chemotherapy alone. This multifunctional platform may enable efficient, safe and precise monitoring and theranostic therapy in the biomedical field. Methods Materials. The chemicals used in this study were bismuth nitrate pentahydrate (Bi(NO3)3·5H2O, ≥ 99.99+%, Aladdin), sodium selenite (Na2SeO3, ≥ 97.0%, Shenyang Huadong reagent), hydroxylamine (NH2OH, 50 wt.% in H2O, Sigma-Aldrich), polyvinylpyrrolidone (PVP, Mw ≈ 55,000, Sigma-Aldrich), acetone ( ≥ 99.9%, Aladdin), ethylene glycol (EG > 99%, Aladdin), dopamine hydrochloride (≥ 98%, Sigma-Aldrich),

doxorubicin

hydrochloride

(DOX-HCl,

98%,

Aladdin),

tris(hydroxymethyl)

aminomethane (Tris, ≥ 99.9%, Aladdin), human serum albumin (HSA, 96-99%, Shanghai Shifeng Biological Technology Co., LTD), dimethyl sulfoxide (DMSO, > 99%, Aladdin), Cell Counting Kit-8 (CCK8, Dojindo Laboratories), calcein acetoxymethyl ester (Calcein AM, > 90.0%, Dojindo Laboratories), PI (Dojindo Laboratories) and DAPI (Dojindo Laboratories). Unless otherwise stated, all the chemicals and reagents were of analytical grade and used as received. Deionized (DI) water with a resistivity of 18.2 MΩ⋅cm was from Milli-Q Water Purification System. Instruments. The morphology, particle size and thickness of the NPs were characterized by SEM (SU8020, Hitachi Limited, Japan), TEM (Tecnai G20, FEI Co., USA) and AFM (Multimode 8, Bruker, USA), respectively. Energy dispersive spectroscopy (EDS) was used to analyze the elements in the


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Bi2Se3 sample. The dynamic light scattering (DLS) and zeta potential of the NPs were analyzed by 90Plus/BI-MAS instrument (Brookhaven Instruments Co., USA). The absorption and infrared (IR) spectra were collected using ultraviolet-visible-NIR spectro-photometer (UV-vis-NIR, Evolution 300, Thermo Scientific, USA) and IR spectrometer (Nicolet 6700, Thermo Scientific, USA), respectively. The NIR irradiation was performed with a continuous-wave diode laser with a center wavelength of 808 ± 10 nm and an output power of 2 W (Beijing Kaipulin Optoelectronic Technology Co., China). The solution temperature was measured via a thermocouple microprobe (STPC-510P, Xiamen Baidewo Technology Co., China). Drug release was determined by using fluorescent spectrophotometer (Cary Eclipse, Varian, USA). The cellular uptake efficiency was studied by a fluorescent inverted microscope (IX71, Olympus, Japan). Synthesis of Bi2Se3@PDA/DOX/HSA NPs. Bi2Se3 cores were synthesized according to the method published previously.17-18 In brief, 0.25 g PVP was dissolved in 40.0 mL ethylene glycol, and 60 mg Na2SeO3 and 112.5 mg Bi(NO3)3⋅5H2O were added in under magnetic stirring at room temperature (RT). Then, the mixture was heated to 180°C in nitrogen environment. Subsequently, the reaction was triggered by rapid injection of 0.6 mL of hydroxylamine solution. 10 min later, the solution was cooled down to RT. The final products were precipitated by centrifuging and washed three times with a mixture of acetone and DI water. Finally, the precipitate was dried in an oven under vacuum at 50°C for 12 h. The PDA coating was performed similar to the method described by Lee et al.40 24.76 mg dopamine hydrochloride was dissolved in 10.0 mL of Tris-HCl (pH = 8.5) solution with an addition of 5.0 mg Bi2Se3 powder. 6 h later, the reaction solution was centrifuged and washed using DI water. The final coating was performed using a modified DA-plus-HSA method to load both HSA and DOX in a single mixed layer.21-22 10.0 mg DOX powder was dissolved in 1.0 mL of DMSO and mixed with 2.0 mL of Bi2Se3@PDA solution (in DMSO). After stirring for 10 min, the mixture was added dropwise into 12.0 mL of 5.0 mg/mL HSA solution with sonication. The obtained product was centrifuged and washed with DI water. Finally, the precipitate was re-dispersed in PBS and stored at 4°C before use.


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Photothermal experiments. The temperature rise induced by NIR laser irradiation was measured by monitoring the temperature of Bi2Se3@PDA/DOX/HSA NP dispersions in DMEM containing 10 % FBS at various concentrations (0, 5, 10, 20, 50, 100, 200 µg/mL) irradiated by a NIR laser (808 nm, 1.2 W/cm2). 3.0 mL of Bi2Se3@PDA/DOX/HSA NP dispersion in a quartz cuvettes (total volume of 4.0 mL) was irradiated by the 808 nm laser for 10 min. The temperature of the solution was measured every 10 s by a thermo-couple microprobe submerged in the solution. Meanwhile, the temperature of 3.0 mL of DMEM containing 10% FBS under radiation was measured as a control. CT signal measurement. 0.5 mg/mL Bi2Se3@PDA/DOX/HSA NPs dispersion (in DI water) was treated with a strong oxidizing mixture containing 1.0 mL of 65% nitric acid and 200.0 mL of H2O2, and then heated to 120°C for 1 h to remove all organics. The remaining inorganic material was dissolved in 10.0 mL of 2% dilute nitric acid medium and analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Calibration curves were adopted for Bi using standard solutions (ICP Element Standard solutions, Merck). Solutions of Bi2Se3@PDA/DOX/HSA NPs suspended in PBS at different Bi concentrations (0, 4.6, 13.7, 27.4, 36.5, 45.6 mmol/L) were prepared in 1.5 mL eppendorf tubes and swirled for 2 min before CT imaging. CT scans were performed using a GE Light Speed VCT imaging system (GE Medical Systems) operated at 100 kV and 80 mA, with a slice thickness of 0.625 mm. Contrast enhancement was calculated in Hounsfield units by averaging over the 3D-based region of interest for each sample. Drug release in vitro. Bi2Se3@PDA/DOX/HSA NPs were suspended in PBS at different pH, sealed in a Float-A-Lyzer○R G2 dialysis tube (approximate molecular weight cutoff 8,000-10,000 Da, Spectrum Laboratories Inc.) and then immersed into 80.0 mL of the same PBS at 37.0°C under moderate shaking. The releasing rate of equivalent free DOX sealed in the dialysis tube was also investigated. At predetermined time intervals, 2.0 mL of the release medium was withdrawn from exterior solution and replaced with an equal volume of fresh PBS. The amount of released DOX at each time point was determined by its fluorescence spectrum using the corresponding standard calibration curve. ACS Paragon Plus Environment

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Cellular uptake and internalization. To study the cellular uptake of Bi2Se3@PDA/DOX/HSA NPs, HeLa and HUVEC cells were incubated with 50 µg/mL Bi2Se3@PDA/DOX/HSA NP dispersions for 2h or 4h at 37.0°C. Thereafter, the cells were immediately fixed and then characterized using fluorescence microscope. In a separate experiment, HeLa cells were irradiated with the NIR laser (808 nm, 1.2 W/cm2) for 5 min after incubation with 50 µg/mL Bi2Se3@PDA/DOX/HSA NP dispersions for 2 h. Then, the cells were rinsed with PBS and characterized using fluorescence microscope. The effect of chemotherapy in vitro. HeLa cells were first seeded in 24-well cell-culture plates and then incubated with Bi2Se3 and Bi2Se3@PDA/DOX/HSA at selected concentrations for 2, 12, and 24h, respectively. Afterwards, the cells were washed with PBS for microscopic observation. To further evaluate the cell survival efficiency after treatment, HeLa cells were first seeded in 96-well cell-culture plates at a density of 1 × 104 cells per well. After incubation for 24h, the cells were exposed to free DOX, Bi2Se3, Bi2Se3@PDA/HSA NPs or Bi2Se3@PDA/DOX/HSA NPs at selected concentrations and incubated for another 24h or 48h. Cell survival efficiency was measured using the CCK-8 assay. The data represented the mean of triplicate measurements. The cells cultured with the pure medium were used as a control. DAPI staining for nuclear morphology study. HeLa cells (1 × 105 cells per well) were first seeded in 12-well cell-culture plates, then incubated with Bi2Se3 (without DOX loading, 20 µg/mL), free DOX, and Bi2Se3@PDA/DOX/HSA NPs (0, 5, 10, 15, and 20 µg/mL) for 24 h at 37.0°C. Afterwards, the cells were fixed with 4% paraformaldehyde for 20 min, and stained with 2 µmol/L DAPI for 15 min. Then, the cells were washed with PBS (pH 7.4) and examined using fluorescence microscope. Investigation of photothermal effect on cancer cells. The localized photothermal cell toxicity of the Bi2Se3@PDA/DOX/HSA NPs was evaluated on HeLa cells. For qualitative analysis, HeLa cells were seeded on a 24-well plate at a density of 2.5 × 105 cells per well at 37.0°C in a humid atmosphere containing 5% CO2. After overnight incubation, Bi2Se3@PDA/DOX/HSA NP dispersion (1.0 mL per well, 50.0 µg/mL in DMEM containing 10% FBS) were added, and then the cells were exposed ACS Paragon Plus Environment

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immediately to NIR laser (808 nm, 1.2 W/cm2) for 0 min, 5 min and 10 min, respectively. After laser irradiation, the cells were incubated with fresh DMEM containing 10% FBS at 37.0°C for 30 min, and then washed with PBS and stained with calcein AM (2.0 µmol/L) and PI (3.0 µmol/L) for visualization of the live cells and dead cells. To further evaluate the cell survival efficiency after radiation, HeLa cells were first seeded on a 96-well plate at a density of 1 × 104 cells per well at 37.0°C in a 5% CO2 atmosphere. After overnight incubation, the cell culture medium was removed and the cells were washed three times with PBS to get rid of dead cells, followed by addition of different concentrations of Bi2Se3@PDA/DOX/HSA NP dispersions (0 µg/mL to 40.0 µg/mL in DMEM containing 10% FBS) at 37.0°C. Then the cells were immediately irradiated with an NIR laser (808 nm, 1.2 W/cm2) for 0 min, 5 min and 10 min, respectively. Cell viability was measured using the CCK-8 assay according to the manufacturer suggested procedures. Results were shown as mean ± standard deviation (SD) (n = 3). Thermo-chemotherapy in vitro. To further evaluate the cell survival efficiency after drug and/or laser treatment and study the synergistic effect for killing cancer cells through combined photothermal therapy and chemotherapy of Bi2Se3@PDA/DOX/HSA NPs under NIR irradiation, HeLa cells were seeded on a 96-well plate at a density of 1 × 104 cells per well. After overnight incubation at 37.0°C in a 5% CO2 atmosphere, the cell culture medium was discarded and the cells were washed three times with PBS to remove dead cells, followed by incubation with gradient concentrated free DOX, Bi2Se3@PDA/HSA or Bi2Se3@PDA/DOX/HSA NPs dispersions at 37.0°C for 12h, respectively. Thereafter, the drug dispersions were replaced with fresh DMEM containing 10% FBS and the cells were irradiated with a NIR laser (808 nm, 1.2 W/cm2) for 5 min. After laser irradiation, the cells were further incubated at 37.0°C for 24 hours. Finally, CCK-8 assay was used to measure the cell viability. Blood circulation and biodistribution in vivo. To measure the blood circulation, 10-15 µL blood was drawn from the untreated side of tail vein in tumor-bearing Balb/c mice at different time-intervals after injection of Bi2Se3@PDA/DOX/HSA (~ 0.81 mg/kg in terms of DOX, and ~ 25 mg/kg in terms of Bi2Se3). Then, the blood samples were weighted and dissolved in HNO3/H2O2 solution (HNO3:H2O2 = ACS Paragon Plus Environment

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2:1) for digestion overnight. The obtained homogeneous solution was diluted and analyzed by ICP-OES. For biodistribution study, tumor-bearing Balb/c mice (three per group) were sacrificed at 2 h, 12 h and 24 h respectively after i.v. injection of Bi2Se3@PDA/DOX/HSA (in terms of DOX ~ 0.81 mg/kg, and in terms of Bi2Se3 ~ 25 mg/kg). Organs and tissues were collected, weighed and then solubilized by HNO3/H2O2 solution in flasks overnight. Then, the flasks were heated to at 120 °C for 2 h to remove all organics, followed by diluting with 2% HNO3 for ICP-OES measurement of Bi element. The levels of Bi2Se3@PDA/DOX/HSA in blood and organs were presented as the percentage of the injected dose per gram of tissue (% ID/g). Thermo-chemotherapy in vivo. Female BALB/c nude mice (5-7 weeks old) were purchased from Shanghai SLAC laboratory Animal Co., Ltd. (SLAC). All animal experiments were carried out under protocols approved by the Institutional Animal Care and Use Committee. To develop the tumor model, HeLa cells (1×106) suspended in 100 µL of PBS were subcutaneously injected into the back of each mouse. After the tumors grew to 8-10 mm in diameter, the mice were divided into five groups. Each group of mice were intravenously injected with 100 µL of PBS, DOX, Bi2Se3, or Bi2Se3@PDA/DOX/HSA (in terms of DOX ~ 0.81 mg/kg, and in terms of Bi2Se3 ~ 25 mg/kg). After 12 h, the mice were anesthetized with isoflurane and the tumors were treated with or without 808 nm laser (0.64 W/cm2) for 10 min. During the period of treatment, the tumor surface temperatures were recorded by IR thermal imager (Ti25, Fluke, USA). After treatments, the length and width of the tumors were measured by a digital caliper every 2 days. The tumor volume was calculated as follows: V=

ab 2 2

(1)

where V, a and b are the tumor volume (mm3), the tumor length (mm) and tumor width (mm), respectively. Relative tumor growth ratio (G) was calculated as follows: G(%) =

V × 100% V0


(2)

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where V and V0 are the tumor volume on day 8 and on day 0, respectively. The percentage of the tumor growth inhibition (TGIR) was calculated according to the following equation:

 G  × 100% TGIR(%) =  1 − G0  

(3)

Histology analysis and blood analysis in vivo: Necropsy was performed on the mice from the treatment group and control group. Major organs (include liver, spleen, kidney, heart, and lung) were placed in 4% paraformaldehyde solution, embedded in paraffin, and cryo-sectioned into 4 µm slices. The frozen slides were further stained with hematoxylin & eosin (H&E) and examined using fluorescence microscope with a digital camera. For serum biochemistry assay and complete blood panel test, 1 mL of blood was collected from each mouse.

Conflict of Interest: The authors declare no competing financial interest. Supporting Information Available: Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. *Address correspondence to [email protected], [email protected], [email protected] Author Contributions: The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Acknowledgement: This work is financially supported by the National Natural Science Foundation of China (Grant No. 21473045, No. 51401066), the Fundamental Research Funds from the Central University (Grant No. HIT.BRETIII.201216, HIT.BRETIII. 201225, HIT.BRETIV. 201313 and PIRS OF HIT A201503), and State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. 2015TS06). M.Y. acknowledges financial support by the Recruitment


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Program of Global Experts, China.

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TOC 140x98mm (300 x 300 DPI)


Multifunctional Bismuth Selenide Nanocomposites for Antitumor

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