Ultrasmall Semimetal Nanoparticles of Bismuth for Dual-Modal

Apr 10, 2017 - School of Radiation Medicine and Protection (SRMP) and School of Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Inn...
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Ultrasmall Semimetal Nanoparticles of Bismuth for Dual-Modal Computed Tomography/Photoacoustic Imaging and Synergistic Thermoradiotherapy Xujiang Yu,† Ang Li,† Chengzhi Zhao,† Kai Yang,*,‡ Xiaoyuan Chen,§ and Wanwan Li*,† †

State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China ‡ School of Radiation Medicine and Protection (SRMP) and School of Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, Jiangsu 215123, People’s Republic of China § Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland 20892, United States S Supporting Information *

ABSTRACT: Multifunctional nanomaterials with integrated diagnostic and therapeutic functions, combination therapy to enhance treatment efficacy, as well as low toxicity have drawn tremendous attentions. Herein, we report a multifunctional theranostic agent based on peptide (LyP-1)-labeled ultrasmall semimetal nanoparticles of bismuth (Bi-LyP-1 NPs). Ultrasmall Bi NPs (3.6 nm) were facilely synthesized using oleylamine as the reducing agent and exhibited a higher tumor accumulation after being conjugated with the tumor-homing peptide LyP-1. The abilities to absorb both ionizing radiation and the second near-infrared (NIR-II) window laser radiation ensured that Bi-LyP-1 NPs are capable of dual-modal computed tomography/photoacoustic imaging and efficient synergistic NIR-II photothermal/radiotherapy of tumors. Moreover, Bi-LyP-1 NPs could be rapidly cleared from mice through both renal and fecal clearance and almost completely cleared after 30 days. Such multifunctional nanoparticles as efficient cancer theranostic agents, coupled with fast clearance and low toxicity, shed light on the future use of semimetal nanoparticles for biomedicine. KEYWORDS: semimetal nanoparticles, bismuth, the second near-infrared (NIR-II) window, multimodal imaging, thermoradiotherapy use in the clinic.15−17 Moreover, synergistic therapeutic approaches,3,18 in addition to multimodal imaging provided by a single system,19−21 are critical for the development of highly efficient cancer theranostics. Radiotherapy (RT) is an extensively used approach to treat cancer in clinical medicine. However, detrimental effects on normal tissues22 and the hypoxic nature23 of tumors remain the two primary issues with this treatment option that limit its efficacy and applications. Recently, nanomaterials with high Zelements (e.g., gold,24,25 iodine,26 bismuth,27 and rare earth elements28,29) have been demonstrated to function as excellent radiosensitizers that are able to concentrate higher radiation

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ith the advanced progress achieved in nanotechnology and biomedicine, various multifunctional nanomaterials has been exploited for uses in cancer diagnosis and therapy.1−4 The ability to embrace multiple modalities in a single nanostructure or one particle (referred to as nanotheranostics)5,6 enables multifunctional nanomaterials to provide uses in cancer imaging, simultaneous diagnosis, and individualized treatment.7 Various multifunctional nanomaterials that have been developed based on intelligent designs to combine different functional nanoparticles8−10 or to utilize certain individual nanoparticles11,12 for multiple applications have made great progress in the field of nanomedicine. However, because there is a risk of toxicity that could be induced by the accumulation of nanomaterials in the body, the use of nanomaterials for further biomedical applications may be limited.13,14 Thus, nanomaterials with smaller size that allow for rapid clearance from the body would be of significant value for © 2017 American Chemical Society

Received: January 21, 2017 Accepted: April 10, 2017 Published: April 10, 2017 3990

DOI: 10.1021/acsnano.7b00476 ACS Nano 2017, 11, 3990−4001

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Figure 1. Scheme and characterization. (a) Scheme of multifunctional Bi NPs. (b) TEM images and HRTEM images of as-synthesized Bi NPs. Inset histogram shows the measured diameters of as-synthesized Bi NPs. (c) XRD pattern of as-synthesized Bi NPs. (d) Absorbance spectrum of PEGylated Bi NPs. (e) Temperature changes of PEGylated Bi NPs at different concentrations under a 5 min irradiation from a 1064 nm laser at 1W cm−2. (f) Temperature profile of a PEGylated Bi NP solution (120 ppm of Bi) under heating with the laser on and then cooling with the laser off. (g) Calculation of the time constant for heat transfer using a linear regression of the cooling profile. (h) Temperature variations of a PEGylated Bi NP solution (200 ppm of Bi) over six cycles of heating and natural cooling.

doses within tumors,30 thus enhancing RT efficacy while reducing possible side effects associated with the treatment.31 Meanwhile, approaches that can be utilized with RT to increase cancer treatment efficacy have also been explored.32−34 Interestingly, mild hyperthermia has been demonstrated to improve oxygen levels within tumors by increasing the blood flow, thus enhancing the tumor killing ability of this treatment when used in conjunction with radiotherapy.23,35,36 Photothermal therapy (PTT) is a well-known treatment used in cases of hyperthermia. This treatment works through the use of nanomaterials with high near-infrared (NIR) absorbance properties that enable them to generate heat to induce tumor ablation under laser irradiation.37 This treatment is minimally invasive and possesses a high therapeutic efficacy, enabling photothermal therapy to be applied either alone or in combination with other treatments,38,39 such as radiotherapy.35 The use of a second near-infrared (NIR-II, 1000−1350 nm) window laser, particularly within 1000−1100 nm, allows deeper

penetration and higher maximum permissible exposure (MPE),40 which could dramatically increase photothermal efficacy. However, prior research has focused primarily on employing a first near-infrared (NIR-I, 700−950 nm) window laser as the excitation light source.41,42 This is likely due to the strong absorption band within the NIR-I window, but negligible absorption within the NIR-II window of traditional nanomaterials (e.g., organic compounds,43 gold nanostructures,44,45 or nanocarbons46,47). Fortunately, increased attention devoted to this area has enabled the exploration of several kinds of nanomaterials as potential photothermal nanomaterials that respond to the NIR-II window, including transition metal sulfide/oxide semiconductors,48−51 noble metal nanomaterials,52−54 and polymer nanocomposites.55 By carefully tuning doping manners or adjusting stoichiometric ratios, semiconductors can exhibit very different optical band gaps and free carrier concentrations, characteristics that strongly contribute to semiconductor absorption in the near-infrared 3991

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ACS Nano region.56 It has recently been reported that semimetal nanomaterials of antimony exhibit both broad and strong absorption characteristics in the range of 700−1350 nm.57 This could potentially be due to their higher charge carriers compared to semiconductors, which increases their value as agents for photothermal therapy in NIR windows. Another typical semimetal element, bismuth (Bi), has attracted much interest owing to its properties (e.g., small carrier effective masses, a long Fermi wavelength, and a small band overlap energy). The recent discovery of plasmonic properties that originate from the semimetal-to-semiconductor transition (also called nanoconfinement effects) in bismuth at the nanoscale58 has broadened the possible applications of Bibased nanomaterials (traditionally used as thermoelectric materials, catalysts, and sensors59) for use in biomedicine as cancer therapeutic agents.21 Moreover, research suggests that the Bi element could also function as a radiosensitizer and a contrast agent for computed tomography (CT).60,61 These potential biomedical applications motivated us to develop ultrasmall bismuth nanoparticles to be used as multifunctional agents for efficient cancer theranostics. To our best knowledge, no studies concerning both photothermal and radiation properties of Bi nanoparticles, their use in multimodal imaging, and their further development for biomedical applications have been reported so far. Herein, a theranostic agent was developed based on the semimetal nanoparticles of Bi, which exhibit strong absorption properties in the NIR-II window, suitable for both dual-modal CT/photoacoustic (PA) imaging and the synergistic PTT/RT treatment of tumors (Figure 1a). First, a facile method for ultrasmall Bi nanoparticles was proposed using oleylamine (OAm) as both the reducing and the stabilizing agent. PEGylated Bi nanoparticles (NPs) were found to exhibit a broad absorption from ultraviolet to NIR region and were capable of acting as an excellent photothermal agent when irradiated with a 1064 nm laser, which possessed high photothermal conversion efficiency and photostability. Once labeled with a peptide (CGNKRTRGC, LyP-1), the resulting nanoparticles (Bi-LyP-1 NPs) were able to present both high cellular uptake and tumor accumulation and also exhibited a potential for use as a CT and PA imaging contrast agent in vivo, due to the absorption ability of both X-ray and NIR light of Bi. Strong synergistic effects were also observed on tumor-bearing mice when Bi-LyP-1 NPs were used in combination with PTT and RT. It should be noted that Bi-LyP-1 NPs exhibited excellent in vivo biocompatibilities and low long-term toxicity and were found to be rapidly cleared via both liver and renal clearance mechanisms with stable morphology. The successful demonstration of utilizing Bi nanoparticles as efficient agents for cancer diagnostics and therapy from our findings sheds light on the future use of Bi nanoparticles and other multifunctional nanomaterials based on semimetals for cancer theranostics.

interplanar d spacing of 0.139 nm was revealed by highresolution transmission electron microscopy (HRTEM) (Figure 1b). The characteristic X-ray powder diffraction (XRD) pattern further demonstrated the presence of Bi NPs with a pure rhombohedral phase (JCPDS No. 44-1246) (Figure 1c). This facile method, which used the common chemicals bismuth acetate, oleic acid, and oleylamine, exhibited great advantages over other reported methods that required precursors specially prepared through multiple steps,62,63 such as Bi[N(SiMe3)2]3. Next, the as-synthesized nanoparticles were mixed with 1,2-diastearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)] (molecular weight 5000, DSPE-PEG5000) containing 15% maleimide terminal groups to yield hydrophilic PEGylated Bi NPs. In order to improve tissue specificity, PEGylated Bi NPs were labeled with a cyclic peptide (CGNKRTRGC, LyP-1), which possesses the ability to specifically target p32 proteins expressed by 4T1 mouse breast cancer cells64 through a thioether reaction65 between the maleimide and the N-terminal cysteine moiety. The resulting nanoparticles (Bi-LyP-1 NPs) were found to be conjugated with approximately 3.6 peptides on each nanoparticle (Figure S1). Bi-LyP-1 NPs showed a hydrodynamic size of about 12 nm and exhibited excellent stability in different physiological solutions (Figure S2a,b). The surface modification induced a negligible change in the morphology of the Bi NPs as monitored by TEM (Figure S2c). Moreover, the successful coupling of DSPE-PEG5000 and conjugation of LyP-1 peptides on the Bi NPs were confirmed by the FTIR spectra and NMR spectra. The intensities of both C−H and C−O stretch bands (2896 and 1101 cm−1) increased distinctly after coupling, which were the two intense peaks of DSPE-PEG5000 (Figure S3a,b), and further studies on the NMR spectrum of PEGylated Bi NPs demonstrated the existence of malemide (6.69 ppm) and methoxy (3.37 ppm) groups on the surface of Bi NPs (Figure S4a). The appearance of a broader and stronger N−H stretch band (3351 cm−1), as well as a stronger CO stretch band (1667 cm−1) in the FTIR spectrum of Bi-LyP-1 NPs, was attributed to 10 amino groups in each peptide (Figure S3c), indicating the conjugation of LyP-1 peptide to Bi NPs, which could also be inferred from the disappearance of the maleimide peak and the appearance of an amino hydrogen peak at 2.85 ppm in the NMR spectrum of Bi-LyP-1 NPs (Figure S4b).66 In Vitro Photothermal Performance. The PEGylated Bi NPs exhibited a strong, broad absorbance band between 200 and 1100 nm, with no obvious sharp peaks (Figure 1d), and this observation prompted us to use the common 1064 nm laser as a source to trigger NIR-II photothermal therapy. Next, we evaluated the photothermal properties of PEGylated Bi NPs by testing the temperature variation of each nanoparticle solution with different concentrations under 1064 nm laser irradiation. We found that when irradiated at a fixed power density, the photothermal heating effect of PEGylated Bi NPs was concentration-dependent (Figure 1e). The solution temperature was increased by about 17.2 °C at 200 ppm of Bi, while the temperature increase of pure water was only about 2.8 °C. Photothermal conversion efficiency (η) is an important parameter used to assess photothermal agents. The η value in our work was calculated to be 32.2% with 1064 nm laser irradiation (Figure 1f,g). This was calculated according to the method established by Roper and co-workers67 and indicates good photothermal properties of PEGylated Bi NPs. When the concentration was fixed (200 ppm of Bi), the temperature rise was dependent on the power density of the laser, with a

RESULTS AND DISCUSSION Synthesis and Characterization of Bi-LyP-1 NPs. Inorganic Bi nanoparticles were first synthesized through a facile organic protocol. Upon injection of the bismuth precursor into hot oleylamine under nitrogen, Bi3+ was gradually reduced by the mild reducibility of oleylamine into ultrasmall nanoparticles and were stabilized by oleylamine molecules as well. Following a 10 min incubation at 260 °C, high-crystalline semimetal Bi NPs were yielded with a diameter of about 3.6 nm, and the clear lattice fringe of Bi NPs with the 3992

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Figure 2. Cell experiments, blood circulation, and biodistribution of Bi-LyP-1 NPs in mice. (a) Relative cell viabilities were evaluated with a CCK-8 assay. Both 4T1 cells and L02 cells were incubated with different concentrations of Bi-LyP-1 NPs for 24 h. (b) Cellular uptake of BiLyP-1 NPs and PEGylated Bi NPs by 4T1 cells following a 6 h incubation. (c) Inhibitory effect of Bi-LyP-1 NPs (0 or 10 ppm of Bi) on 4T1 cells under various irradiation doses. (d) Blood circulation of Bi-LyP-1 NPs in mice by detecting the percentage of Bi remaining in the blood among injected dose at different time points. (e) Biodistribution of Bi-LyP-1 NPs and PEGylated Bi NPs in mice after 24 h of circulation. (f) Calculated ratios of Bi-LyP-1 NPs relative to PEGylated Bi NPs in the main tissues (*p < 0.05, **p < 0.01).

variation of approximately 9.2 °C obtained at 0.6 W cm−2 (Figure S5a,b). Photothermal stability of Bi nanoparticles was examined further with a 5 min, 1064 nm laser irradiation of 1 mL of PEGylated Bi NP solution (200 ppm of Bi) at 1 W cm−2 and then leaving it to cool to environmental temperature. The above cycle was repeated for a total of six times, and the consistency of the temperature changes was observed (Figure 1h), proving the good photostability properties of PEGylated Bi NPs upon irradiation. This observation was further supported by TEM images, XRD pattern, and absorption spectrum following the six-cycle irradiation of PEGylated Bi NPs (Figure S6). In Vitro Cytotoxicity Assay. Prior to the use of Bi-LyP-1 NPs for tumor targeting and image-guided thermoradiotherapy, we investigated the interactions of nanoparticles with cells. A standard cell counting kit-8 (CCK-8) assay was used to test the cytotoxicity of Bi-LyP-1 NPs after different concentrations of the Bi-LyP-1 NP solution were incubated with 4T1 cancer cells and hepatic L02 cells for 24 h, respectively (Figure 2a). No distinct cytotoxicity to both cancer and normal cells was observed after 24 h of incubation with the Bi-LyP-1 NPs in our tested dose range. It is noteworthy that the relative viabilities of 4T1 cells (81%) was slightly lower than that of hepatic L02 cells (90%) at 400 μg mL−1, which could be attributed to the higher concentration of Bi-LyP-1 NPs accumulated in 4T1 cells than in L02 cells owing to the highly expressed p32 proteins (the receptor of LyP-1) of 4T1 cancer cells compared to L02 normal cells.61 The 48 h relative viabilities of 4T1 cells and L02 cells incubated with Bi-LyP-1 NP solution at the same concentrations were also investigated, and a similar result of low cytotoxicity was obtained (Figure S7). In Vitro Cellular Uptake. Cellular uptakes of peptidelabeled and nonlabeled Bi NPs were then measured to quantify

cellular uptake efficacy of NPs enhanced by LyP-1 peptides after internalized with 4T1 cancer cells for different internalization times. As shown in Figure 2b and Figure S8, the uptake efficacy of Bi-LyP-1 NPs increased gradually before reaching saturation at about 6 h, while that of nonlabeled Bi stayed nearly unchanged after about 5 h. The uptake efficacy of LyP-1labeled Bi NPs was greater than that of nonlabeled Bi NPs at each time point, with an approximately 2.4-fold greater cellular uptake after 6 h of incubation. Such saturable and timedependent uptake behaviors by 4T1 cells indicated the cell endocytosis of nanoparticles, and the additional cellular uptake of LyP-1-labeled Bi NPs was attributed to the homing specificity of LyP-1 peptides to the highly expressed p32 receptors at the cell surface.61 These results indicate an enhanced specificity of LyP-1-labeled Bi NPs for 4T1 cancer cells. In Vitro Radiation Therapy. Upon demonstrating the capability of Bi NPs to trigger PTT, we carried out an in vitro clonogenic survival assay to determine whether Bi NPs had the ability to enhance RT efficacy. After being cultured in the presence or absence of Bi-LyP-1 NPs for 24 h, 4T1 cells were irradiated by X-ray at 0, 2, 4, 6, and 8 Gy, respectively. The results of the clonogenic survival assay (Figure 2c) showed BiLyP-1 NPs (10 ppm of Bi) could remarkably enhance RT efficacy, and the inhibitory effect was dependent on X-ray irradiation doses. Owing to the excellent photoelectric effect and Compton scattering of Bi, the surviving fraction (SF) of cells treated with nanoparticles at 4 Gy was found to be approximately 0.107, while the SF of the untreated cells was 0.219. Survival fractions were also fitted by a single-target model formula (Table 1), with the SF2 under 2 Gy X-ray irradiation calculated to be 0.602 for untreated 4T1 cells and 0.415 for cells treated with Bi-LyP-1 NPs (10 ppm of Bi). The 3993

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significant radiosensitization effect, even at a low concentration and a low dose of radiation. In Vivo Blood Circulation. Motivated by these exciting in vitro experimental results, we moved forward to carry out in vivo studies. Specifically, the pharmacokinetic behavior of Bi-LyP-1 NPs in mice was studied in order to determine the blood circulation half-life. For these studies, mice were intravenously (i.v.) injected with Bi-LyP-1 NPs (3 mg mL−1, 200 μL), followed by collecting blood samples at nine time points in order to measure the amount of Bi by inductively coupled plasma mass spectrometry (ICP-MS). The half-life of NPs circulating in the blood was determined to be approximately 3.97 h (Figure 2d). The blood content of Bi-LyP-1 NPs was found to remain steady at a relatively low level following 24 h of circulation. This could be due to the ultrasmall size and surface modifications of Bi NPs.

Table 1. Calculated Parameter Values and Fitted Formula Based on the Radiosensitization Activity of Bi-LyP-1 NPs in 4T1 Cells D0

Dq

SF2

X-ray

1.550

1.628

0.602

Bi-LyP-1 + X-ray

1.273

1.230

0.415

SERD0

1.218

SER10

1.248

fitted formula SF = 1 − (1 − e−0.645D)2.858 SF = 1 − (1 − e−0.786D)2.637

sensitizing enhancement ratio of SERD0 was calculated to be 1.218 for 4T1 cells treated with Bi-LyP-1 NPs (10 ppm of Bi), which was similar to that of the reported WS2 quantum dots while with a much higher concentration (100 ppm of W) (1.22).68 Moreover, the SER10 of Bi-LyP-1 NPs reached up to 1.248, and this value was higher than that of Au NPs (1.19).25,34 It is clear that Bi-LyP-1 NPs demonstrate a

Figure 3. Dual-modal CT and PA imaging both in vitro and in vivo with Bi-LyP-1 NPs. (a) HU values of various concentrations of Bi-LyP-1 NPs and iohexol solutions. (b) X-ray CT images of Bi-LyP-1 NPs and iohexol solutions of corresponding concentrations. (c) X-ray CT images of tumors obtained prior to and 5 min after administration of Bi-LyP-1 NPs. (d) PA images and (e) PA signal intensities of tumors obtained before and after administration of Bi-LyP-1 NPs at 700 nm. (f) PA images and (g) PA signal intensities of tumors obtained at each time point after administration of Bi-LyP-1 NPs at 700 nm. (h) Photograph of the experimental mice for PA imaging taken at 8 h post i.v. injection. (**p < 0.01). 3994

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Figure 4. Combined treatment of tumors with Bi-LyP-1 NPs. (a) IR images of mice under laser irradiation after administrated with Bi-LyP-1 NPs and PBS, respectively. (b) Corresponding temperature variation of tumors in (a). (c) Recorded curves on tumor growth in each group. (d) Photographs of tumors in each group after 14 days. (e) H&E (top) and CD31 staining (bottom) of tumor sections under different treatments (**p < 0.01, ***p < 0.005).

In Vivo Tumor Accumulation. In addition, the biodistribution of both the peptide-labeled and nonlabeled Bi NPs (3 mg mL−1, 200 μL) was studied in 4T1 tumor-bearing mice at 24 h following administration (Figure 2e). Both Bi NPs accumulated at high levels in the spleen, liver, and kidneys, while at rather low levels in the lungs and heart. A similar tissue distribution pattern was observed for both nanoparticles in these organs (Figure 2f), indicating no obvious influence of the peptide modification on distribution in mice. However, Bi-LyP1 NPs did exhibit a remarkable 1.7-fold enhancement in tumor accumulation (6.26 ± 0.33% ID/g) in contrast to that of PEGylated Bi NPs (3.66 ± 0.48% ID/g). These results correlate with those obtained in in vitro cellular experiments, demonstrating the successful modification of peptide LyP-1 on nanoparticles and the clear enhancement of tissue specificity observed with Bi-LyP-1 NPs. In Vitro and in Vivo X-ray Computed Tomography. Prior to using Bi-LyP-1 NPs for cancer treatment, we aimed to understand their efficacy as dual-modal CT and PA imaging agents on mice. Although bismuth has been proposed as an excellent CT contrast element, and several Bi-based nanoparticles have been synthesized for this reason,64,69−71 no

research examining Bi nanoparticles alone has yet to be reported. We first studied the in vitro Hounsfield units (HU value) of Bi-LyP-1 NPs in phosphate-buffered saline (PBS) (pH = 7.4), with iohexol used as a control (Figure 3a,b). The results revealed that Bi exhibited a significantly higher slope of HU value (approximately 13.8 HU mM−1) than iohexol (approximately 4.28 HU mM−1). These results indicate the excellent CT signal enhancement capability of Bi-LyP-1 NPs. We next carried out further studies aimed at investigating in vivo CT imaging (Figure 3c). Here, CT imaging were conducted on mice with intratumoral (i.t.) injection of BiLyP-1 NPs (6 mg mL−1, 30 μL) both prior to and 5 min after administration. Upon injection of the nanoparticles, an obvious tumor contrast was observed, with the highest increase of HU value from 130.5 to 1193.6. This observed increase suggests that Bi-LyP-1 NPs alone could be utilized as highly effective CT contrast agents. In Vivo Photoacoustic Imaging. As a widely used, noninvasive biomedical modality, PA imaging employs laser light to acoustically visualize biological tissues. This technique is advantageous in that it can provide high imaging depth and spatial resolution.72 Because the properties of Bi nanoparticles 3995

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Figure 5. In vivo behavior and long-term toxicity of Bi-LyP-1 NPs. (a) Long-term biodistribution of Bi-LyP-1 NPs in main organs of mice at each time point after administration. (b) Detected amount of Bi in feces and urine at each time point after i.v. injection. (c) TEM images of feces collected at 24 h after i.v. injection. Inset shows a magnified view. (d) Blood biochemistry of mice sacrificed at each time point, including alanine minotransferase (ALT), alkaline phosphatase (ALP), amino transferase (AST), blood urea nitrogen (BUN), albumin/globin ratios (A/ G), and total protein (TP). (e) Hematology data contained eight blood panel parameters of mice sacrificed at each time point.

of a 700 nm laser source. Similar results were also observed under 800 and 900 nm laser irradiation (Figure S9a−c). These results not only coincided with a high accumulation of Bi-LyP-1 NPs in tumors (Figure 3h) but also indicated the great promise Bi NPs hold as highly efficient contrast agents for PA imagingguided cancer treatment under a wide range of NIR wavelengths. NIR-II Photothermal and Radiotherapy in Vivo. After successfully demonstrating the photothermal properties of NIR-II, we utilized RT enhancement and dual-modal imaging of Bi-LyP-1 NPs for further studies. The in vivo PTT and RT

enable a strong and broad NIR absorption, we conducted PA imaging in mice administrated with Bi-LyP-1 NPs (6 mg mL−1, 30 μL) via i.t. injection at different wavelengths. A high PA signal intensity was observed in tumors in mice following the i.t. injection, with an increase of approximately 3.3-fold when exposed to 700 nm laser irradiation (Figure 3d,e). PA imaging in mice administrated with Bi-LyP-1 NPs (6 mg mL−1, 200 μL) via i.v. injection was carried out at various time points postadministration (Figure 3f,g). From these studies, the PA signal intensity of tumors was found to be increased approximately 3.6-fold at 8 h post i.v. injection under irradiation 3996

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obtained in tumor-bearing mice (Figure 2e). The levels of BiLyP-1 NPs remaining in these organs was found to decrease sharply at 7 d postinjection, with very low Bi levels detected after 30 days. The results from these studies indicate that BiLyP-1 NPs can be almost completely cleared from mice. In order to gain further insights into the clearance routes of nanoparticles, urine and feces were collected from mice that had received an i.v. injection of Bi-LyP-1 NPs (3 mg mL−1, 200 μL). From these samples, Bi levels were measured using ICPMS. The fact that Bi was found to be present in both urine and feces (Figure 5b) suggests that these nanoparticles are cleared through fecal excretion and renal clearance mechanisms. Such clearance mechanisms would align with the observation of a high accumulation of Bi-LyP-1 NPs in the spleen, liver, and kidney. Differences in the amount of Bi in feces and urine with time could suggest that the hydrodynamic size plays a role in clearance. It has been reported that nanoparticles greater than 10 nm in size have a tendency to accumulate in RES organs, while those with diameters smaller than 5.5 nm can be easily cleared by the kidneys due to the fact that the effective pore size of the renal clearance barrier is approximately 8 nm.27,73 Because the hydrodynamic size of Bi-LyP-1 NPs is in the range of 8−18 nm, we can infer that nanoparticles greater than 10 nm in size are absorbed by the liver and spleen for slow, long-term excretion. Nanoparticles in the range of 8−10 nm in size are likely to accumulate in the kidney, where they are rapidly cleared via renal filtration. From the variation in the trend of the amount of Bi detected in urine and feces, it is likely that kidney clearance is the primary clearance mechanism on the first day, with fecal excretion gradually taking over as the primary clearance route. This could be explained by a decrease in renal clearance capacity, caused by the gradual accumulation of nanoparticles in the kidney, which could diminish the permeability of the glomerular basement membrane and podocytes.74 In addition, Bi NPs in urine and feces that were excreted after 24 h were characterized by TEM (Figure 5c and Figure S12). These studies show essentially no change in the subspherical morphology of Bi NPs, further indicating the excellent stability properties of Bi-LyP-1 NPs in mice. Further investigations aimed to demonstrate the low long-term toxicology of Bi-LyP-1 NPs at a 2-fold treatment dose (6 mg mL−1, 200 μL) were also carried out. These studies included serum biochemistry and a complete blood panel measurement. As depicted in Figure 5d,e, the serum biochemistry results showed the presence of liver and kidney function markers, while the parameters of complete blood tests were found to be within normal ranges at each time point after administration. In addition, H&E staining of main organs showed no visible inflammation or damage was induced to treated mice compared to untreated mice (Figure S13). With these results, we confirmed that Bi-LyP-1 NPs possess excellent in vivo biocompatibility and low long-term toxicity.

combined treatment of a subcutaneous mouse tumor model was used to study six different treatment groups. Using IR imaging (Figure 4a), we first studied the surface temperature of tumors in mice with i.v. injection of Bi-LyP-1 NPs (3 mg mL−1, 200 μL) that were irradiated by the 1064 nm laser at 0.6 W cm−2. We showed that the surface temperature of these tumors rapidly increased to approximately 45 °C and then stabilized, while that of tumors injected with PBS was found to slowly increase to approximately 39.5 °C (Figure 4b). However, we found that this mild photothermal treatment alone in mice injected with Bi-LyP-1 NPs resulted only in a delay in the growth of tumors (Figure 4c). Similar results were also observed in mice exposed to X-ray treatment, with a preinjection of either PBS or Bi-LyP-1 NPs. In these cases, tumors from both treatment groups were observed to continue to gradually grow during the rest time points due to the lowdose X-ray treatment. We demonstrate that when combined together with Bi-LyP-1 NPs, X-ray irradiation was capable of inhibiting the tumor growth more effectively (Figure 4c). These results clearly demonstrate the ability of Bi-LyP-1 NPs to enhance RT efficacy. In remarkable contrast to the limited therapeutic effect observed for PTT alone (Bi-LyP-1 NPs with laser) or RT alone (Bi-LyP-1 NPs without laser), impressive results on the inhibition of tumor growth were observed when RT and PTT treatments were combined. As shown in Figure 4c,d, the growth of tumors in mice i.v. injected with Bi-LyP-1 NPs was completely inhibited following exposure to both a 1064 nm wavelength laser and X-ray irradiation. These results clearly indicate the positive synergistic effect of PTT and RT therapies on the inhibition of tumor growth. In order to better elucidate changes in the internal structures of tumors that underwent different treatment regimens, we performed hematoxylin and eosin (H&E) staining, as well as CD31 staining, in tumor slices derived from these experimental groups (Figure 4e). Both significant levels of cell apoptosis and efficient suppression of angiogenesis in tumors were observed only in the PTT and RT combination treatment group. These results correlate with the effective therapeutic results observed in these treatment groups. It should also be noted that excellent therapeutic results were obtained in the case of tumors treated with only 0.6 W cm−2 for the 1064 nm laser excited PTT and a low dose of 4 Gy for RT. This treatment regimen may lower levels of pain and other side effects throughout the course of the treatment. Moreover, all mice were observed to behave normally (Figure S10), with no death or significant body weight changes observed (Figure S11) throughout the course of treatment. The work presented here demonstrates not only the use of Bi NPs as excellent agents for NIR-II photothermal and radiotherapy treatments but also the exhibited synergistic effects observed on the targeting of tumors when these therapies are utilized together. Blood Panels, Histology Examinations, and Clearance Study in Vivo. Because both the distribution and clearance of NPs, as well as their potential long-term toxicity in vivo, are crucial for their development in further biomedical applications, we investigated these properties of Bi-LyP-1 NPs in mice. For these studies, mice i.v. injected with Bi-LyP-1 NPs were sacrificed at 1, 7, and 30 days following administration (n = 5) to measure the amount of Bi in major organs (spleen, liver, kidney, lung, and heart). The major organs in which Bi was found to accumulate were the spleen and liver, followed by the kidney, while the heart and lungs showed relatively low levels of Bi accumulation (Figure 5a), which agreed with the results

CONCLUSION In summary, we show that ultrasmall Bi nanoparticles with diameters of approximately 3.6 nm could be facilely synthesized using oleylamine as the stabilizing and reducing agent. As demonstrated by the strong absorbance in the second NIR window, Bi nanoparticles were found to exhibit excellent NIRII photothermal properties, as well as superior photostability. Following the modification of the tumor-homing peptide LyP1, the cellular uptake efficacy and tumor accumulation of BiLyP-1 NPs were about 2.4-fold and 1.7-fold higher than those 3997

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Quantification of the Number of Peptides on Each Nanoparticle. To determine the density of peptides on each nanoparticle, LyP-1-(fam) peptide that was labeled with the 5(6)-carboxyfluorescein (fam) dye was purchased (95.5% purity, GL Biochem Ltd. China) and then conjugated on PEGylated Bi NPs with the same protocols used for LyP-1 peptides. The 5(6)-carboxyfluorescein exhibited a characteristic absorption peak at 497 nm and could be used to quantify the peptide concentration, while the numbers of nanoparticles could be calculated with the data of average diameter from TEM, crystal parameters of the XRD pattern, and the concentration of Bi measured with ICP-MS. The typical protocol for LyP-1-(fam) peptide conjugation was kept the same as that of LyP-1 peptides: 1 mg of as-synthesized Bi NPs was first reacted with 8.5 mg of DSPE-PEG5000methoxy and 1.5 mg of DSPE-PEG5000-maleimide and then purified with 100 kDa ultrafiltration filters before redispersed in 3 mL of PBS to conjugate with 3 mL of 200 μM LyP-1-(fam) peptides at 25 °C for 4 h. After purification with 0.2 μm PVDF filters to remove possible agglomerates, washing with ice-cold PBS solution, and centrifugation with 100 kDa ultrafiltration filters several times to get rid of dissociative peptides, a concentrated solution of 1 mL was obtained, with a total of 10 mL of filtrated solution containing nonlabeled LyP-1(fam) peptides. The concentration of Bi was measured with ICP-MS, and the concentration of nonlabeled LyP-1-(fam) peptides was measured using a UV−visible−NIR spectrophotometer. In Vitro Photothermal Performance. To measure photothermal transduction, 1 mL of PEGylated Bi NP aqueous dispersion of various concentrations was added into quartz cuvettes and then irradiated using a 1064 nm laser source. In vitro temperature variation videos and thermal images versus time were recorded using the E50 camera. In Vitro Cytotoxicity Assay and Cellular Uptake. Both 4T1 cancer cells and hepatic L02 cells (7 × 103 cells/well) were grown in 96-well plates under standard conditions (37 °C, 5% CO2) with the culture medium of 90% RPMI 1640 medium and 10% fetal bovine serum (FBS, Sigma-Aldrich) for 24 h, followed by replacement with complete medium containing a series of Bi-LyP-1 NPs and incubation for another 24 and 48 h. The cells were washed with fresh medium three times before CCK-8 assays were performed to assess the viabilities. To determine the ability of 4T1 cells to internalize Bi nanoparticles and to compare the specific binding affinity of Bi-LyP-1 NPs versus nonlabeled Bi NPs, 4T1 cells (1 × 105 cells/well) were grown in sixwell plates for 24 h. After replacing the culture medium with complete medium containing 50 ppm Bi-LyP-1 NPs, the plates were then incubated at 37 °C for a series of times (0.5, 2, 4, 6, and 12 h). Following this incubation, 4T1 cells were washed with medium three times, released with cell dissociation buffer (Invitrogen, USA), and centrifuged. The resulting pellet was then treated with concentrated aqueous HNO3 for 12 h. The amount of Bi was then analyzed using ICP-MS. Control samples were performed at the same time in both of the above experiments. Animal Experiments. Six-week-old Balb/c mice were managed under protocols approved by Soochow University Laboratory Animal Center. Tumors of mice were prepared with injection of 60 μL of PBS containing 2 × 106 4T1 cells into the fat pad of their right legs, and tumor sizes were calculated with the following equation: volume (V) = length × width2/2. In Vivo Blood Circulation. A 200 μL amount of Bi-LyP-1 NP solution (6 mg mL−1) was i.v. injected into healthy mice (n = 5), and blood samples were collected by eyeball removal at 5 min and 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h postinjection. These samples were treated with concentrated aqueous HNO3, and the amount of Bi was measured using ICP-MS. In Vitro Radiation Therapy. In order to evaluate the radiosensitization effect of Bi-LyP-1 NPs, 4T1 cells were initially plated into six-well plates (100, 200, 500, 1000, and 2000 cells/well) for 24 h, followed by replacing the culture medium with complete medium containing Bi-LyP-1 NPs (0 or 10 ppm) for another 24 h of incubation. After being irradiated with different X-ray irradiation doses (0, 2, 4, 6, 8 Gy), the cells were then further incubated in fresh medium for 10 days under standard conditions. Following this 10-day

of nonlabeled Bi NPs, respectively. These simple nanostructures (Bi-LyP-1 NPs) exhibited excellent potential as candidate agents for dual-modal CT/PA imaging. Additionally, the capability of Bi-LyP-1 NPs to absorb both ionizing radiation and NIR-II laser radiation, as well their ability to enhance RT efficacy, provides numerous beneficial properties to these nanoparticles. We show that these nanoparticles are able to produce a remarkable, effective synergistic effect of PTT and RT on tumor treatment, even irradiated at 0.6 W cm−2 and 4 Gy, while PTT or RT treatment alone was found to only partly inhibit the growth rate of tumors. Studies on the long-term toxicity and clearance behaviors of Bi-LyP-1 NPs in mice revealed that these nanoparticles are cleared through both renal and fecal clearance mechanisms, with nanoparticles almost completely cleared after 30 days, with no damage or death observed in mice. Such a multifunctional theranostic agent with excellent imaging and therapeutic properties, coupled with its fast clearance and low long-term toxicity, demonstrates great promise for the use of semimetal nanoparticles in the field of biomedical science.

MATERIALS AND METHODS Materials. Oleic acid and oleylamine were purchased from Aladdin. 1,2-Diastearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (molecular weight 5000, DSPE-PEG5000) was purchased from Nanocs Incorporation. Bismuth(III) acetate was purchased from Sigma-Aldrich. Characterization. TEM images was recorded with a Tecnai G2 electron microscope (Spirit Biotwin, USA), and HRTEM images were obtained with a JEM-2100F emission microscope (Japan). XRD pattern was performed using a SmartLab (Cu Kα, λ = 1.5418 Å, Japan). FTIR spectra were acquired using a Fourier transform infrared spectrometer (EQUINOX55, Germany). NMR spectra were performed with a Bruker AVANCE III 600 MHz (Germany). ICP-MS analysis of Bi was conducted on an Agilent 7500a Series (USA). Optical absorbance of Bi nanoparticles was measured using a UV− visible−NIR spectrophotometer (EV300, USA). The 1064 nm laser source for photothermal experiments was generated by a laser diode controller (SFOLT. Co. Ltd., China), and thermal images were recorded by an E50 thermal imaging camera (FLIR, USA). Hydrodynamic diameter was tested with a Zetasizer Nano ZS instrument (Malvern, UK). PA imaging was performed using a Vevo LAZR PA system (Vevo LAZR, VisualSonics, Canada). CT imaging was taken on the eXplore Locus (GE, USA). Synthesis of Ultrasmall Bi Nanoparticles. Typically, 1 mmol of bismuth(III) acetate was dissolved in 5 mL of oleic acid under vacuum at 100 °C for 1 h, followed by an additional 30 min under dry nitrogen. This precursor solution was then injected into 25 mL of hot oleylamine at 260 °C, with an entire reaction time of 10 min. Following the reaction, chloroform and ethanol were added, followed by centrifugation of the nanoparticles three times. The final product was stored in chloroform for further use. Synthesis of Bi-LyP-1 Nanoparticles. A cyclic nine amino acid peptide with an N-terminal cysteine moiety (GL Biochem Ltd., 95% purity) was used without further purification. The as-synthesized Bi nanoparticles were coated with DSPE-PEG5000 first (PEGylated Bi NPs) by mixing 1 mg of Bi nanoparticles with 8.5 mg of DSPEPEG5000-methoxy and 1.5 mg of DSPE-PEG5000-maleimide in 3 mL of chloroform prior to labeling with the LyP-1 peptide. The solution was stirred and then evaporated under dry nitrogen, followed by centrifugation with 100 kDa ultrafiltration filters to remove the excess polymers or empty micelles. The resultant dried powder was dissolved in PBS (pH = 7.4) under sonication at 70 °C for 5 min. Once the powder was redispersed, 3 mL of 200 μM LyP-1 solution was immediately added and reacted at 25 °C for 4 h with moderate agitation. The reaction product was purified through a 0.2 μm PVDF filter and a 100 kDa ultrafiltration filter and then stored for further use. 3998

DOI: 10.1021/acsnano.7b00476 ACS Nano 2017, 11, 3990−4001

Article

ACS Nano period, the surviving colonies were counted (only when more than 50 cells), and each treatment group was repeated for a total of three times. Surviving fraction = (surviving colonies)/(cells seeded × plating efficiency). The sensitizer enhancement ratios SERD0 and SER10 that describe the ratios of X-ray irradiation doses needed to kill 63% and 90% of the total cells were calculated by the equation SER = D(control group)/D(experimental group), the fitted formula of SF = 1 − (1 − e−D/D0)n, and parameters of mean lethal dose (D0) and quasi-threshold dose (Dq) based on practical experimental data. In Vivo Tumor Accumulation. Tumor-bearing mice and healthy mice were given an i.v. administration of 200 μL of Bi-LyP-1 NP solution (3 mg mL−1) and euthanized after 24 h, respectively (n = 5). Tumors and main organs were digested with concentrated HNO3 overnight in order to analyze the amount of Bi in each tissue using ICP-MS. Dual-Modal Imaging. For in vitro CT imaging, Bi-LyP-1 NP solutions and iohexol solution containing various concentrations of Bi and I were detected using the CT instrument. For in vivo CT imaging, 30 μL of Bi-LyP-1 NP solution (6 mg mL−1) was administrated into tumors in mice, and CT imaging was performed both prior to and after administration, with parameters of 80 kV, 450 μA, and a slice thickness of 45 μm. As for in vivo PA imaging, experimental mice were adminstrated with 30 μL of Bi-LyP-1 NP solution (6 mg mL−1) via i.t. injection and 200 μL of Bi-LyP-1 NP solution (6 mg mL−1) via i.v. injection, respectively. PA imaging was carried out both prior to and after each injection. Throughout the entire course, mice were anesthetized and body temperature was maintained around 37.5 °C. NIR-II Photothermal and Radiotherapy in Vivo. Six randomly divided groups of mice were employed for therapy: (I) control, (II) BiLyP-1 NPs, (III) X-ray, (IV) Bi-LyP-1 NPs + laser, (V) Bi-LyP-1 NPs + X-ray, and (VI) Bi-LyP-1 NPs + laser + X-ray. Once the tumors attained ca. 100 mm3, mice in each group were administrated with 200 μL solution of PBS (groups I and III) or Bi-LyP-1 NPs (3 mg mL−1, the other four groups) via i.v. injection, respectively. After blood circulation for 24 h, group V was treated with laser irradiation (1064 nm, 0.6 W cm−2, 20 min), groups III and IV were treated with X-ray irradiation (4 Gy), and group VI was treated with both laser irradiation and X-ray irradiation (1064 nm, 0.6 W cm−2, 20 min, 4 Gy). During each laser irradiation treatment, the temperature profiles were captured in real time using a thermal camera. Following each treatment, H&E and CD31 staining and tumor slice analysis were performed. The data of body weight and tumor volumes in each group over the course of 14 days were recorded. In Vivo Clearance Study. For excretion studies, five mice that were i.v. administrated with 200 μL of Bi-LyP-1 NP solution (3 mg mL−1) were raised in metabolic cages for 7 days. Each day, urine and feces samples were collected for further detection of Bi with TEM and ICP-MS. For long-term biodistribution studies, healthy mice that were i.v. administrated with 200 μL of Bi-LyP-1 NP solution (3 mg mL−1) were scarified at day(s) 1, 7, and 30 after administration (n = 5), and main organs were collected to analyze Bi levels using ICP-MS. Blood Panels and Histology Examinations. Five healthy mice that received an i.v. administration of Bi-LyP-1 NPs at a 2-fold treatment dose (6 mg mL−1, 200 μL) were euthanatized at day(s) 1, 7, and 30 after administration. Five additional mice i.v. administrated with PBS were used as a control. Standard procedures for serum biochemistry and a complete blood panel measurement were performed with blood obtained from mice at the different time points. In addition, the heart, liver, spleen, lung, and kidneys were collected for H&E staining. Images were collected using an inverted laboratory microscope (Leica DM IL LED). Statistical Analysis. The data were analyzed using a one-way ANOVA statistical analysis and indicated with p-values for significance.

Figures S1−S13 (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xujiang Yu: 0000-0002-8916-0742 Xiaoyuan Chen: 0000-0002-9622-0870 Wanwan Li: 0000-0003-3809-0737 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Project Nos. 81371645, 81471716, 31400861, 81671782), Science and Technology Committee of Shanghai (Project Nos. 15PJD020, 15441905800, 16JC1400604), the Natural Science Foundation of Jiangsu Province (BK20140320), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Medicine & Engineering Cross Research Foundation of Shanghai Jiao Tong University (Project No. YG2014MS33), and the Intramural Research Program, National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH). We thank the Instrumental Analysis Center of SJTU for the assistance with TEM, XRD, XPS, and ICP-MS characterizations. REFERENCES (1) Melancon, M. P.; Zhou, M.; Li, C. Cancer Theranostics with Near-Infrared Light-Activatable Multimodal Nanoparticles. Acc. Chem. Res. 2011, 44, 947−956. (2) Yoo, D.; Lee, J.-H.; Shin, T.-H.; Cheon, J. Theranostic Magnetic Nanoparticles. Acc. Chem. Res. 2011, 44, 863−874. (3) Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat. Rev. Cancer 2005, 5, 161−171. (4) Lim, E.-K.; Kim, T.; Paik, S.; Haam, S.; Huh, Y.-M.; Lee, K. Nanomaterials for Theranostics: Recent Advances and Future Challenges. Chem. Rev. 2015, 115, 327−394. (5) Lammers, T.; Aime, S.; Hennink, W. E.; Storm, G.; Kiessling, F. Theranostic Nanomedicine. Acc. Chem. Res. 2011, 44, 1029−1038. (6) Muthu, M. S.; Leong, D. T.; Mei, L.; Feng, S.-S. Nanotheranostics-Application and Further Development of Nanomedicine Strategies for Advanced Theranostics. Theranostics 2014, 4, 660−677. (7) Barreto, J. A.; O’Malley, W.; Kubeil, M.; Graham, B.; Stephan, H.; Spiccia, L. Nanomaterials: Applications in Cancer Imaging and Therapy. Adv. Mater. 2011, 23, H18−H40. (8) Ambrogio, M. W.; Thomas, C. R.; Zhao, Y.-L.; Zink, J. I.; Stoddart, J. F. Mechanized Silica Nanoparticles: A New Frontier in Theranostic Nanomedicine. Acc. Chem. Res. 2011, 44, 903−913. (9) Ng, K. K.; Lovell, J. F.; Zheng, G. Lipoprotein-Inspired Nanoparticles for Cancer Theranostics. Acc. Chem. Res. 2011, 44, 1105−1113. (10) Song, G.; Wang, Q.; Wang, Y.; Lv, G.; Li, C.; Zou, R.; Chen, Z.; Qin, Z.; Huo, K.; Hu, R.; Hu, J. A Low-Toxic Multifunctional Nanoplatform Based on Cu9S5@mSiO2 Core-Shell Nanocomposites: Combining Photothermal- and Chemotherapies with Infrared Thermal Imaging for Cancer Treatment. Adv. Funct. Mater. 2013, 23, 4281− 4292. (11) Chen, J.; Wang, D.; Xi, J.; Au, L.; Siekkinen, A.; Warsen, A.; Li, Z.-Y.; Zhang, H.; Xia, Y.; Li, X. Immuno Gold Nanocages with Tailored Optical Properties for Targeted Photothermal Destruction of Cancer Cells. Nano Lett. 2007, 7, 1318−1322.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00476. 3999

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DOI: 10.1021/acsnano.7b00476 ACS Nano 2017, 11, 3990−4001

Article

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DOI: 10.1021/acsnano.7b00476 ACS Nano 2017, 11, 3990−4001