DTX Multiple Nanorods for

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Biological and Medical Applications of Materials and Interfaces

Laser Induced Transformable BiS@HSA/DTX Multiple Nanorods for Photoacoustic/Computed Tomography Dual-Modal Imaging Guided Photothermal/Chemo Combinatorial Anticancer Therapy Weisheng Guo, Jing Chen, Lu Liu, Ahmed Shaker Eltahan, Nicola Rosato, Jing Yu, Dongliang Wang, Jingqi Chen, Massimo Bottini, and Xing-Jie Liang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16395 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 10, 2018

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ACS Applied Materials & Interfaces

Laser Induced Transformable BiS@HSA/DTX Multiple Nanorods for Photoacoustic/Computed Tomography Dual-Modal Imaging Guided Photothermal/Chemo Combinatorial Anticancer Therapy Weisheng Guo,*,†,# Jing Chen,

‡,#

Lu Liu,§ Ahmed Shaker Eltahan,† Nicola Rosato, ⊥ Jing Yu,‖

Dongliang Wang,§ Jingqi Chen,*,† Massimo Bottini,§,⊥ and Xing-Jie Liang* ,§ † Translational Medicine Center, State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou 510260, China; ‡ School of Life Sciences, Tianjin University, Tianjin 300072, China; § Laboratory of Controllable Nanopharmaceuticals, Chinese Academy of Sciences (CAS) Center for Excellence in Nanoscience and CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China; ⊥Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome 00133, Italy; ‖ College of Materials Science and Engineering, Zhejiang University of Technology, Hangzhou 310014, China # These authors contribute equally

Corresponding Authors: Weisheng Guo: [email protected]; Jingqi Chen: [email protected]; Xing-Jie Liang: [email protected]; Keywords: Photoacoustic Imaging, Photothermal Therapy, Structure Transformation, Nanomedicines, Drug Release

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Abstract Sub-optimal intra-tumor accumulation and poorly controllable release of encapsulated drugs remain unresolved challenges hampering further advancement of nanomedicines on cancer therapy. Herein, we conceived near infrared (NIR) laser-triggered transformable BiS@HSA/DTX multiple nanorods (mNRs), which were made of small bundles of bismuth sulfide nanorods (BiS NRs) coated by docetaxel (DTX)-inlaid human serum albumin (HSA). The BiS@HSA/DTX mNRs had a lateral size of approximately 100 nm and efficiently accumulated in the tumor microenvironment upon systemic administration in tumor-bearing nude mice. NIR laser irradiation of the tumor area caused rapid disassembly of the BiS@HSA/DTX mNRs into individual HSA-coated BiS nanorods (BiS@HSA iNRs) and triggered the release of DTX from the HSA corona, due to the local temperature increase generated by BiS NRs via photothermal effect. The laser-induced transformation into BiS@HSA iNRs facilitated their penetration and increased the retention time in tumor. The spatiotemporal delivery behavior of the BiS@HSA/DTX mNRs could be monitored by photoacoustic/computed tomography dual-modal imaging in vivo. Furthermore, because of the excellent photothermal conversion properties of BiS NRs and laser-triggered DTX release from BiS@HSA/DTX mNRs, efficient tumor combinatorial therapy was achieved via concurrent hyperthermia and chemotherapy in mice treated with BiS@HSA/DTX mNRs upon NIR laser irradiation.


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1. Introduction Over the past decades, nanomedicines (NMs) have emerged as promising tools to improve the therapeutic index of conventional anti-cancer drugs. This property is largely attributable to the preferential accumulation of NMs in tumors by exploiting the enhanced permeability of angiogenic blood vessels and dysfunctional lymphatic drainage of tumor tissues, phenomena collectively referred to as the enhanced permeability and retention (EPR) effect.1, 2 Enormous technological progress has been made in this field, however, a major obstacle to the clinical translation of NMs stems from their sub-optimal intra-tumor accumulation and penetration in the tumor deep zones.3 Upon systemic administration in the body, NMs encounter numerous sequential biological barriers, including opsonization in the bloodstream - a process that signals liver Kupffer cells to remove foreign bodies from the bloodstream - and low pH, high pressure and/or hypoxia in the tumor microenvironment.3 These barriers may hamper the extravasation of the nanoparticles from the tumor vessels as well as their penetration in the deep zones of the tumor. Another obstacle to the translation of NMs into clinics arises from the poor control of release of encapsulated drugs. Liposome-encapsulated Cisplatin SPI77 showed greater intra-tumor accumulation than free Cisplatin, however it failed a phase III clinical trial due to the poor drug bioavailability upon drug release.4, 5 Thus, the design of innovative NMs is urgently needed to avoid these obstacles and achieve a significant increase in tumor killing with respect to current approaches based on free drugs. Increasing evidence has shown that the intra-tumor trafficking profile of systemicallyadministered NMs is associated with their physicochemical properties, including size,6, 7 shape,8 and surface coating.9, 10 In particular, size has a crucial impact on nanoparticle accumulation, penetration, and retention at the tumor site.11-13 Nanoparticles with an average size of ~100 nm display long blood circulation time and efficient extravasation from tumor vessels, however they may suffer from poor penetration in the deep zones of the tumor because of diffusional hindrance.14-16 On the contrary, nanoparticles with an average size smaller than 25 nm can permeate more deeply in the tumor and 3 ACS Paragon Plus Environment


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display a more uniform intra-tumor distribution and longer retention time than larger counterparts, but may suffer from insufficient tumor accumulation.17, 18 To overcome this “size dilemma” and develop efficacious cancer therapies, researchers have designed various size-adjustable NMs, which possess larger initial sizes in the bloodstream for slower clearance and efficient extravasation from tumor vessels and transform into smaller particles within the tumor region in response to internal and/or external stimuli for better intra-tumor distribution and longer retention.19-24 These NMs could also offer spatiotemporal control of drug bioavailability by enabling “on-demand” drug release.22,

25-28

For

instance, reports have described NMs displaying “on demand” size change and/or drug release by cleavage of chemical bonds triggered by ultraviolet (UV) light,25 acidic pH,27, 29, 30 or enzymes.31, 32 Despite the impressive conceptual advancements, some concerns may arise from the use of hazardous UV light, time-consuming transition response and the presence of residual tags upon chemical bond cleavage. Thus, the development of innovative NMs that can rapidly transform into smaller nanoparticles in response of a stimulus and enable to achieve a precise drug regimen in the tumor still remains an unresolved challenge. Herein, we describe near infrared (NIR) laser-triggered transformable BiS@HSA/DTX multiple nanorods (mNRs) as efficient cancer theranostic tools. As depicted in Scheme 1, small bundles of hydrophobic uniform bismuth sulfide (BiS) NRs were bound together by human serum albumin (HSA), resulting in hydrophilic BiS@HSA mNRs. Docetaxel (DTX) was readily entrapped in the HSA corona due to electrostatic and hydrophobic interactions,33 resulting in BiS@HSA/DTX mNRs. The nanoparticles had an average size of approximately 100 nm and efficiently accumulated in the tumor via EPR effect upon systemic administration in nude mice bearing MDA-MB-231 breast cancer. NIR laser irradiation of the tumor area triggered the disassembly of BiS@HSA/DTX mNRs into individual HSA-coated BiS nanorods (BiS@HSA iNRs) and the release of DTX from the HSA corona due to the local temperature increase generated by BiS NRs via photothermal effect. In virtue of their small size (~40 nm in length), BiS@HSA iNRs penetrated deeper in the tumor and persisted for longer time with 4 ACS Paragon Plus Environment

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respect to BiS@HSA/DTX mNRs. The spatio-temporal trafficking profile of BiS@HSA/DTX mNRs in the tumor could be monitored by photoacoustic (PA)/computed tomography (CT) dual-modal imaging in a non-invasive manner in vivo by exploiting the intrinsic properties of BiS NRs as PA and CT contrast imaging agents. Furthermore, due to the photothermal conversion property of BiS NRs and laser-triggered DTX release, efficient tumor killing was also achieved upon NIR laser irradiation by a combination of photothermal therapy and chemotherapy. Taken together, our BiS@HSA/DTX mNRs hold promising potentials as scaffolds for the fabrication of efficient and controllable cancer theranostic approaches.

2. Results and discussion 2.1. Synthesis and imaging properties of BiS@HSA/DTX mNRs First, monodispersed BiS NRs were synthesized via a well-developed “hot injection method” with BiCl3 and elemental sulfur as precursors (see experimental section in Supporting Information).34 Transmission electron microscopy (TEM) revealed that the BiS NRs had a uniform morphology with a length of 44±5.7 nm and a diameter of 5.3±1.2 nm (Figure 1A). BiS NRs displayed a well-defined crystallinity (Figure 1A). Energy dispersive X-ray (EDX) elemental mapping demonstrated that the S (in red color) and Bi (in green color) elements were uniformly distributed in the NRs (Figure 1B). Next, BiS@HSA/DTX mNRs were fabricated via an ultrasonication-emulsion technique with the addition of DTX (Figure 1C).35, 36 The resutant BiS@HSA/DTX mNRs were made of bundles of BiS NRs, which are kept together with DTX-inlaid HSA by wall-to-wall hydrophobic interactions (Figure 1D and Figure S2A, in Supporting Information). The HSA coating improved the dispersibility of the nanoparticles in aqueous environments.37 As shown in Figure 1D, the BiS NRs were successfully transferred from chloroform to aqueous phase upon coating with the DTX-inlaid HSA, which caused negligible changes in their absorption spectrum. TEM images showed that the BiS@HSA/DTX mNRs had a lateral size of 103±18.6 nm in dehydrated state, whereas dynamic light scattering (DLS) 5 ACS Paragon Plus Environment


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measurements showed that the nanoparticles had a hydrodynamic diameter (HD) of 286±32.6 nm (Figure 2I and Figure S3 in Supporting Information). The BiS@HSA/DTX mNRs were investigated as PA/CT dual-modal imaging contrast agents by embedding the nanoparticles in agar gel cylinders and testing them in a multispectral optoacoutic tomography (MSOT) imaging system. A monotonically decreasing PA signal was recorded in the NIR region, which well corresponded to the optical absorbance spectrum of the BiS@HSA/DTX mNRs (Figure 1E). The PA imaging phantoms were acquired (Figure 1G) and their corresponding PA intensities increased linearly with the concentration of BiS@HSA/DTX mNRs (Figure 1F). The slope of the plot of the PA intensity vs Bi concentration was derived as 26559 (a.u.)/(mg·mL-1), which suggests that our NMs can behave as efficient contrast agents. The CT contrast efficacy of the obtained BiS@HSA/DTX mNRs was also assessed on a small animal CT scanner in vitro. As showed in Figure 1H and Figure 1I, the BiS@HSA/DTX mNRs produced intense contrast enhancement even at low concentrations, and the Hounsfield units (HU) values increased linearly along with Bi concentration. The BiS@HSA/DTX mNRs’ CT contrast efficacy stemmed from the fact that Bi possesses the largest X-ray attenuation coefficient (Bi: 5.74, Au: 5.16, Pt: 4.99, and I: 1.94 cm2 kg-1 at 100 KeV).38, 39 2.2. Laser Induced Transformation and Drug Release of BiS@HSA/DTX mNRs The broad absorbance spectrum of BiS@HSA/DTX mNRs in the NIR region motivated us to explore their photothermal properties and structural transformations upon laser irradiation. To evaluate the photothermal properties, BiS@HSA/DTX mNRs suspensions at various concentrations were irradiated with a nanosecond pulsed laser source (808 nm) with a power density of 1 W/cm2, and the temperature elevation of the suspensions was recorded with an infrared thermal camera (Fluke Ti400). Double distilled water was selected as control. As depicted in Figures 2A-C, the temperature of BiS@HSA/DTX mNRs suspensions increased with the duration of laser irradiation and the concentration of NMs. In detail, the temperature of a suspension of BiS@HSA/DTX mNRs could easily increase to over 60 oC after 10 min of laser irradiation, while a temperature increase of just 3 oC 6 ACS Paragon Plus Environment

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was recorded for pure water under the same conditions. The increase in temperature of BiS@HSA/DTX mNRs suspensions subjected to laser irradiation was also dependent on laser power density (Figures 2D-F). On the basis of the heating-cooling curve in Figure 2H and Figure S4 (in Surporting Information), the photothermal conversion efficiency of BiS@HSA/DTX mNRs was calculated to be 32.4% via the quantification method by Roper.40 Due to the photothermal conversion property of BiS@HSA/DTX mNRs, we investigated their structural transformation upon laser irradiation by means of TEM and dynamic light scattering (DLS). As shown in Figure 2I and Figure S2 (Supporting Information), BiS@HSA/DTX mNRs made of multiple BiS NRs bound together by DTX-inlaid HSA via hydrophobic interaction and having an average size of 103±18.6 nm were readily obtained via an ultrasonication-emulsion method. After NIR laser irradiation (808 nm, 1 W/cm2, 10 min), numerous individual nanostructures were observed with sizes of approximately 40 nm. This result was explained by the fact that the laser irradiation triggered the disassembly of the nanostructures into many smaller ones, as depicted in Figure 2G. Because of the remarkable photoacoustic effect41, 42

of BiS@HSA/DTX mNRs demonstrated in Figure 1, BiS NRs could generate heat vibration and

caurse severe thermal stress inside the nanostructure under the irradiation of nanosecond pulsed laser, which thereby gave rise to the laser-induced structural transformation. DLS results also showed that the hydrodynamic diameter (HDs) changed from 286±32.6 nm to 63.7±16 nm after laser exposure (Figure S3, Supporting Information). The significant structural transformation further motivated us to exploit the potential effects on the release of DTX. As illustrated in Figure 1C, DTX was loaded into the HSA corona through electrostatic interactions, forming BiS@HSA/DTX mNRs with a loading content up to 2.2%. DTX concentrations were quantified by HPLC, as shown in Figure S4 (Supporting Information). Less than 5% of the entrapped DTX was released from the BiS@HSA/DTX mNRs after 18 h of incubation in PBS buffer at 37 oC. The release of DTX from the BiS@HSA/DTX mNRs was greatly boosted at 3 h from 4.2% to 16.1% after 10 min of laser irradiation and the final cumulative release reached up nearly to 25% of the initial entrapped DTX, which was nearly 5 times higher than 7 ACS Paragon Plus Environment


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the amount of drug released from BiS@HSA/DTX mNRs in absence of laser treatment. As a contrast, when the BiS@HSA/DTX mNRs were incubated in an elevated temperature (50 oC) without laser irradiation, the total cumulative release was less than 10 % and burst release of DTX was not found compared with the incubation at 37 oC circumstance. This validated the laser-triggered release of DTX, which was most likely caused by the local heat vibration42 and thermal stress43 of BiS@HSA/DTX mNRs under the irradiation of nanosecond pulsed laser. Taken together, the laser-triggered disassembly of BiS@HSA/DTX mNRs and release of DTX shed the light on the use of these nanoparticles as efficient anti-cancer approaches. 2.3. In Vitro PTT/Chemo- Therapy Test of BiS@HSA/DTX mNRs The in vitro cancer cell killing efficacy of the BiS@HSA/DTX mNRs was assessed on MDAMB-231 breast cancer cells. The MDA-MB-231 cells were incubated with PBS, BiS@HSA NRs, or BiS@HSA/DTX mNRs at equivalent Bi doses, and subjected or not to laser irradiation (808 nm, 0.5 W/cm2, 10 min). After 24 h of incubation, cell viabilities were quantitated using the cell counting kit8 (CCK-8) assay. The treatment with BiS@HSA mNRs without laser irradiation did not lead to any significant cytotoxicity throughout a wide range of Bi concentrations (0-200 μg/mL), whereas the treatments with BiS@HSA mNRs followed by laser irradiation and with BiS@HSA/DTX mNRs without laser irradiation led to similar cytotoxicity in a dose-dependent manner (Figure 3A). Importantly, the treatment with BiS@HSA/DTX mNRs followed by laser irradiation induced the most effective cell death at all the Bi concentrations tested due to the combination of laser-triggered hyperthermia and DTX release (Figure 2J). Confocal fluorescence images of cells co-stained with propidium iodide (PI) and Calcine AM were also acquired to validate the cell killing efficacy of BiS@HSA/DTX mNRs (Figure 3B). In agreement with the above quantitative results, almost all the cells treated with BiS@HSA/DTX mNRs and irradiated with a NIR laser source displayed a strong red fluorescence (indicative of apoptotic cells), whereas the other treatment groups still exhibited green fluorescence-emitting cells (indicative of viable cells). Furthermore, flow cytometry was employed to 8 ACS Paragon Plus Environment

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assess the ability of BiS@HSA-DTX mNRs to induce apoptosis in cells subjected or not to laser irradiation. As shown in Figure 3C, laser irradiation or incubation with BiS@HSA mNRs alone induced apoptosis in less than 10 % of the cells. Incubation with BiS@HSA/DTX mNRs alone and incubation with BiS@HSA mNRs followed by laser irradiation caused a late apoptosis in 35.3 % and 40.1 %, respectively, of the cells. Finally, treatment with BiS@HSA/DTX mNRs followed by laser irradiation resulted in the highest ratio of late apoptotic cells up to 70.9 %. Collectively, the results of the in vitro experiments confirmed the efficient cell killing ability of BiS@HSA-DTX mNRs through a combination of PTT and chemotherapy. 2.4. In Vivo Tumor-Site Trafficking of the BiS@HSA/DTX mNRs Upon Laser Irradiation Given the favorable in vitro results, we investigated the structural changes and the intra-tumor trafficking of the BiS@HSA/DTX mNRs in vivo by PA and CT dual-modal imaging methodology. PA imaging is an emerging hybrid modality that efficiently overcomes the resolution and penetration limits of optical imaging,44-46 thus enabling to visualize physiological and pathological features of tissues at the molecular level.47-49 To track the intra-tumor trafficking of BiS@HSA/DTX mNRs by PA imaging, 100 μL of a 3 mg/mL BiS@HSA/DTX mNRs solution was intravenously (i.v.) injected into nude mice bearing a MDA-MB-231 breast cancer. Twelve hours post-injection (p.i.), the mice were randomly divided into 2 groups (group I and group II, 3 mice each group). The mice of group I were subjected to laser irradiation (808 nm, 1 W/cm2, 10 min) at 12 h post-injection (p.i.), whereas the mice of group II were not irradiated (control). Cross-sectional PA signals of the tumor area were acquired at various p.i. time-points. As shown in Figure 4A and Figure 4B, the intensity of the PA signal arising from the tumor area was markedly increased 12 h after treatment with BiS@HSA/DTX mNRs. The phenomenon suggested a continuous accumulation of BiS@HSA/DTX mNRs in the tumor due to the efficient exploitation of the EPR effect. Notably, the intensity of the PA signal arising from the tumor area remained steady during the 36 h following the laser irradiation in mice of groups I, whereas it gradually decreased in mice of group II. The orthogonal view of an enlarged tumor area 9 ACS Paragon Plus Environment


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(dotted squares in Figure 4A) showed a uniform PA signal arising from both the periphery and the core of the tumor in mice of group I, whereas a weaker PA signal arising mostly from the periphery of the tumor was observed in mice of group II (Figure 4C). These results suggested that the longer tumor retention of the nanoparticles in irradiated mice (group I) was due to the greater ability of individual BiS@HSA NRs to penetrate deeper in the core of the tumor with respect to aggregated ones. To better assess the role of physical structure on BiS@HSA/DTX mNRs biodistribution, the major organs were collected 48 h after nanoparticle injection and the content of nanoparticles calculated by ICP-MS. As shown in Figure 4D, laser irradiation led to an approximately 2-fold increase in the BiS@HSA/DTX mNRs accumulated in the tumor area with respect to the absence of irradiation (5.9±2.1% ID/g vs 2.8±1.3% ID/g). This phenomenon was accounted to the longer retention and delayed egression out from the tumor of individual BiS@HSA NRs with respect to aggregated ones. To assess the laser-triggered release of a cargo from BiS@HSA/DTX mNRs in vivo, we loaded the nanoparticles with the fluorescent dye Cyanine5.5 (Cy5.5), instead of DTX, resulting in BiS@HSA/Cy5.5 mNRs. As presented in Figure S5 (see Supporting Information), Cy5.5 has a fluorescence emission peak centered at 690 nm, where BiS NRs have an intense absorption, suggesting that the fluorescence of Cy5.5 loaded on BiS@HSA mNRs might be quenched by BiS NRs due to the fluorescence resonance energy transfer effect. Indeed, we found that BiS@HSA/Cy5.5 mNRs had a faint fluorescence signal, whereas a 10-fold increase in fluorescence intensity was obtained upon laser irradiation (808 nm, 1 W/cm2, 10 min) (Figure S5 in Supporting Information). This phenomenon suggested that the changes in fluorescence emission of BiS@HSA/Cy5.5 mNRs can be used as a reporter of cargo release. On the basis of this principle, we injected the BiS@HSA/Cy5.5 mNRs into tumor-bearing mice and exposed the animals to a laser irradiation (808 nm, 1.0 W/cm2, 10 min) 12 h p.i. As shown in Figure 4F and Figure 4G, the mice not subjected to laser irradiation showed a very week fluorescence arising from the tumor area, which might originate from the Cy5.5 diffused out from the nanoparticles. On the contrary, the fluorescence emission from the tumor area increased more 10 ACS Paragon Plus Environment

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than 10-folds and gradually decreased in laser-irradiated mice. Thus, our data suggested the possibility to obtain an accurate spatio-temporal control of drug bioavailability by exploiting the laser-triggered structural changes and drug release of BiS@HSA/DTX mNRs. In light of the CT contrast performance of BiS NRs, 200 μL of BiS@HSA/DTX mNRs solution in PBS (15 mg/mL) were i.v. injected into the tumor bearing mice to perform whole body angiography by CT imaging, when the tumor diameters reached 10 mm. As shown in Figure 4E and Videos S1 and S2 (see Supporting Information), the vasculature was rapidly perfused 15 min p.i. and a clear angiography of heart, kidney, vena jugularis interna, venae subclavia, vena epigastrica, postcaval vein, and tumor microvessels was obtained. The defective and tortuous tumor microvasculature was clearly visible, which sheds the light on the potential use of our nanoparticles for precise cancer theranostics as well as vascular disease diagnosis. 2.5.

In Vivo Chemo/PTT Combination Therapy Evaluation of BiS@HSA/DTX mNRs Due to the efficient delivery and controlled release properties of BiS@HSA/DTX mNRs, we

employed these NMs for PTT/chemo combinatorial cancer therapy in vivo. Mice bearing tumors of approximately 100 mm3 were divided into four groups (5 mice per group) and treated as follow: mice of group I were i.v. injected with PBS (control); mice of group II were treated with a single i.v. injection of BiS@HSA mNRs and irradiated with laser (808 nm, 1 W/cm2, 10 min) 4 h p.i.; mice of group III received 3 i.v. injections of BiS@HSA/DTX mNRs (DTX/body weight = 2.5 mg/kg) every 3 days; mice of group IV received 3 i.v. injections of BiS@HSA/DTX mNRs (DTX/body weight = 2.5 mg/kg) every 3 days and irradiated with laser 4 h after the first injection. Mice of groups II and III exhibited a similar rapid increase in the tumor’s surface temperature to over 45 oC after 10 min of laser exposure (Figure 5A and Figure 5B). The tumors’ sizes were measured every 3 days by using a caliper. As shown in Figure 5C and Figure 5D, mice of group II showed a moderate inhibition of tumor growth (43.1% with respect to control), thus suggesting that the tumors could not be completely destroyed under the mild hyperthermia performed within a safety margin. The triple injection of BiS@HSA/DTX 11 ACS Paragon Plus Environment


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mNRs at a DTX dose of 2.5 mg/kg (DTX/body weight) led to 52.4% of tumor inhibition, which was not statistically different than the tumor inhibition observed in mice of group II. Remarkably, the tumors of mice treated with BiS@HSA/DTX mNRs and irradiated with a NIR laser source were almost completely eliminated with 92.8% of tumor suppression. All the tumors were harvested and weighed at the end of the therapy (Figure 5F). The tumors’ weight confirmed that the treatment with BiS@HSA/DTX mNRs followed by NIR laser irradiation led to the best therapeutic effect due to the combination of PTT and chemotherapy. Furthermore, H&E staining of tumor slices collected 7 days post-treatment showed that most of the cells in the tumors of mice of group IV were severely damaged, while the cells in the tumors of mice of the other treatment groups were only partially destroyed (Figure 5H). The safety and potential toxicity arising from the combinatorial therapy were carefully evaluated by monitoring the body weight of the mice (Figure 5G) and staining histologic slices of the major organs with H&E (Figure 5I). We did not observe neither appreciable body weight loss nor pronounced organ damage in mice of group IV. The blood biochemistry analysis showed that the markers of hepatic (ALT, AST) and renal (CREA, UA, BUN) functions did not differ from those of control mice, which suggested no overt hepatic and renal toxicities due to BiS@HSA/DTX mNRs treatment (Figure S7 in Supporting Information).

3. Experimental Section 3.1. Characterizations The hydrodynamic particle size distributions of the BiS@HSA/DTX mNRs in borate buffer (0.1M, pH=8.5) were measured using the Malvern nanozetasizer (Malvern Instruments, U.K) with an equilibration time of 120 s at 25 oC. The results were analyzed based on three measurements by the Dynamic Nanosizer software. For TEM observation, the BiS NRs in chloroform and BiS@HSA/DTX mNRs in borate buffer were dropped onto 400-mesh copper grids. The morphology of the BiS@HSA/DTX mNRs and BiS NRs was characterized by TEM using a Hitachi (HT7700, operated 12 ACS Paragon Plus Environment

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at 80 kV) transmission electron microscope and a field emission transmission electron microscopy (Tecnai G2 F20 U-TWIN, operated at 200 kV) coupled with energy dispersive X-ray spectroscopy (EDS) analysis, respectively. The UV-vis absorption spectra were recorded on a Hitachi U-3900 spectrophotometer. The real-time thermal imaging and temperature measurement of the quartz was recorded by a Fluke thermal camera (Ti-400). 3.2. Synthesis of BiS Nanorods. BiS nanorods were prepared via a hot-injection method with some developments. Typically, 3.17 mmol of BiCl3 powder was added into a flask containing 4.2 mL of oleylamine followed by degassing at room temperature for 15 min in the existence of argon flow. The solution was slowly heated to 160 °C over the course of 40 min and then maintained at that temperature for 30 min. During the heating process, the mixture turned to be a dark gray solution. Then, 15.85 mmol of sulfur dissolved in 10.4 mL of oleylamine was quickly injected into the flask. The solution changed into red brown upon the injection, and the nanocrystals were allowed to grow for 60 min at 110 °C. The reaction was then terminated with cold hexane. The mixture was centrifuged, the supernatant discarded, and the precipitate was dispersed in toluene. The dispersion was heated to 80 °C in an oven overnight. Unsolubilized materials were then removed by centrifugation, and ethanol was added dropwise to the supernatant until it became turbid. The mixture was centrifuged, the supernatant discarded, and the precipitated nanocrystals redispersed in hexane or toluene. 3.3. BiS@HSA/DTX mNRs Preparation. The BiS@HSA/DTX mNRs were prepared via a developed ultrasonication-emulsion strategy. Typically, 5 mL of water containing 25 mg of HSA was placed under an ultrasonic transducer. Subsequently, 1 mL of BiS nanorods (10 mg) solution in chloroform was added dropwisely into the HSA aqueous solution during ultrasonication (100 W) in 3 min. After ultrasonicating for 8 min, the obtained emulsion mixture was evaporated to remove chloroform and a clear gray aqueous was abtained. The resulted aqueous solution was filtered by a filter (0.22 μm) to remove the large 13 ACS Paragon Plus Environment


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aggregations and centrifuged (10000 g, 15 min) for three times to remove excess HSA. The BiS@HSA mNRs in borate buffer (0.1M, pH=8.5) was obtained and lyophilized for concentration determination by mass. Then 100 μL of acetone containing 1.0 mg of DTX was dropped into 2 mL of the BiS@HSA in borate buffer (10 mg/mL) under stirring. After overnight, the mixture solution was purified by centrifugation (10000 g, 15 min) to remove the free DTX. The obtained BiS@HSA/DTX mNRs were finally dispersed into borate buffer at concentration of 20 mg/mL and kept at 4 oC for further use. 3.4. DTX Release of BiS@HSA/DTX mNRs In Vitro 1 mL of BiS@HSA/DTX mNRs aqueous solution (10 mg/mL) was added into a dialysis tube (3500 Da cut-off membrane), followed by immersion in 10 mL of PBS (1X) containing tween 80 (0.5 %) under mild constant shaking at 37 oC or 50 oC. 0.5 mL of PBS solution was collected at various timepoints. During the dialysis, the PBS solution volume was maintained by refilling with fresh PBS after each sampling. The release experiments were carried out without and with a nanosecond pulsed NIR laser irradiation (808 nm, 1 W/cm2, 10 min). The concentrations of DTX were determined by HPLC with a UV-Vis detector at 230 nm. A mixture of acetonitrile/water (50/50, v/v) was used as the mobile phase. The elution peak of DTX was found to be at 12.23 min. The encapsulation efficiency and loading capacity of DTX in BiS@HSA/mNRs were calculated to be 44% and 2.2%, respectively. 3.5. Cell Line and Animal. Human breast cancer MDA-MB-231 cells were routinely cultured in DMEM medium containing 10% FBS, 100 U/mL streptomycin, and 100 U/mL penicillin (Gibco BRL). The cells were kept in tissue culture flasks in a humidified atmosphere containing 5% CO2 at 37 oC. BALB/c nude mice (~15 g, 6 weeks, female) were purchased from Beijing HFK Bioscience Co., Ltd. The MDA-MB-231 xenograft tumor model was established by injecting 2 million MDA-MB-231 cells in 100 μL of PBS into the right flank of the nude mice. When the tumor volume reached ∼100 mm3, the treatment experiments were conducted in accordance with guidelines approved by the ethics committee of Peking University. 14 ACS Paragon Plus Environment

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3.6. In Vivo MSOT Imaging. BiS@HSA/DTX mNRs (100 μL, 3mg/mL) was I.V. injected into the tumor bearing BALB/c nude mice for photoacoustic imaging, which was acquired on the multispectral optical tomography system (MSOT inVision 128, iThera medical, Germany). Photoacoustic signals were detected under different excitation wavelength (680-900 nm). The two excitation wavelengths were used to measure oxygenated and deoxygenated hemoglobin at 850 nm and 750 nm, respectively. Photoacoustic signals before injection were recorded as control. The orthogonal PA imaging views and ROI photoacoustic signal analysis were performed by the MSOT imaging software. All mice were euthanized after the 48 h imaging. All major organs including tumors were collected for biodistribution quantification by ICPMS based on Bi species concentration determination. 3.7. In Vivo PTT/Chemo Combination Therapy Assessment. When the tumors grown up to approximately 100 mm3, the tumor bearing mice were randomly divided into four groups (5 mice per group) for various treatments: (I) Saline i.v. injection; (II) BiS@HSA mNRs i.v. injection (about 100μL, 15 mg/mL) plus 808nm laser irradiation at 4h p.i. (1W/cm2, 10 min); (III) triple BiS@HSA/DTX mNRs i.v. injections every 3 days (about 100μL, 15 mg/mL; DTX/body 2.5 mg/kg); (IV) triple BiS@HSA/DTX mNRs i.v. injections every 3 days (about 100μL, 15 mg/mL; DTX/body 2.5 mg/kg) plus laser treatment (1W/cm2, 10 min) at 4 h after the first injection. Tumor volume was monitored by measuring the perpendicular diameter of the tumor by a caliper. Tumor volume (mm3) was calculated following the equation: 𝑉 = (𝐿 × 𝑊2) 2 where L and W indicate the length and width of the tumor. The tumor growth inhibition efficiency was calculated based on the following equation:

(

𝑟= 1―

)

𝑉′𝑓 ― 𝑉′0 𝑉𝑓 ― 𝑉0

× 100%



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where 𝑉𝑓 and 𝑉0 represent the final and initial tumor volume of the saline group, while 𝑉′𝑓 and 𝑉′0 represent the final and initial tumor volume of the treatment group.

4. Conclusion In brief, we have successfully constructed BiS@HSA/DTX mNRs that can undergo to NIR lasertriggered structural transformation and be exploited for improved drug delivery and efficacious precise nanotheranostic. The as-prepared BiS@HSA/DTX mNRs displayed a lateral size of approximately 100 nm and readily disassemble in smaller BiS@HSA iNRs with a size of approximately 40 nm in response to NIR laser irradiation due to the photothermal properties of BiS NRs. These NMs not only displayed efficient extravasation from the blood vessels and accumulation in the tumor by exploiting the favorable EPR effect of larger nanoparticles, but also displayed longer retention and better intratumoral distribution by making use of the diffusion superiority of smaller nanoparticles. The intratumor trafficking profile of our NMs could be monitored by PA/CT dual-modal imaging in a real-time and non-invasive manner. Additionally, the entrapped DTX in the BiS@HSA/DTX mNRs could also be readily released due to remarkable photo-thermal effect of the BiS NRs that triggered transformations in the NMs’ structure upon NIR laser irradiation. The features of rapid structure transformation and DTX release in response to NIR irradiation enabled to efficiently eradicate a poorly permeable MDA-MB-231 breast cancer model through a combination of PTT and chemotherapy without any overt systemic toxicity.

Declaration of interest The authors declare no competing financial interest.

Acknowledgments The authors gratefully acknowledge that this work was financially supported by the Chinese National Natural Science Foundation projects (81601603 and 51573128) and National Distinguished Young Scholars grant (31225009). The authors also appreciate the support by the external cooperation 16 ACS Paragon Plus Environment

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program of BIC, Chinese Academy of Science, Grant No. 121D11KYSB20130006 and the "Strategic Priority Research Program" of the Chinese Academy of Sciences Grant No. XDA09030301.

Supporting Information Available Supplementary material (including detailed Experimental Section, TEM images and DLS of BiS@HSA/DTX, Reconstitution videos of the CT imaging, standard curve of DTX concentration versus the integral area under peak at 230 nm derived by HPLC, fluorescence spectrum of BiS@HSA/Cy5.5, and blood biochemistry analysis result) is available.

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Scheme 1. Schematic illustration of the fabrication, structural transformation, and drug release properties of BiS@HSA/DTX mNRs in response to laser irradiation. BiS@HSA/DTX multiple nanorods (mNRs) were made of small bundles of BiS NRs coated by docetaxel (DTX)-inlaid HSA. Upon NIR laser irradiation, BiS@HSA/DTX mNRs disassembled into individual BiS NRs coated by DTX-inlaid HSA (BiS@HSA/DTX iNRs). The BiS@HSA/DTX mNRs were also employed for photoacoustic

(PA)/computed

tomography

(CT)

dual-modal

(PTT)/chemical combinatorial therapy of tumors.


imaging

and

photothermal


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Figure 1. Characterization of BiS@HSA/DTX mNRs. (A) TEM and high-resolution TEM (inset) images of BiS NRs. Inset scale bar = 5 nm. (B) EDX elemental mapping of BiS NRs. (C) Schematic illustration of the fabrication of BiS@HSA/DTX mNRs. (D) UV-vis absorbance spectrum and digital photographs (inset) of hydrophobic BiS NRs in chloroform (red line) and hydrophilic BiS@HSA/DTX mNRs in water (black line). (E) PA signal spectrum of BiS@HSA/DTX mNRs at various concentrations. (F) Linear relationship between PA signal intensity and BiS@HSA/DTX mNRs concentration. (G) PA imaging phantoms of BiS@HSA/DTX mNRs embedded in agar gel cylinders at various concentrations. (H) Linear relationship between Hounsfield units (HU) value and BiS@HSA/DTX mNRs concentration. (I) CT imaging phantoms of BiS@HSA/DTX mNRs suspensions at different concentrations. The BiS@HSA/DTX mNRs concentrations were determined based on the Bi element by ICP-MS.


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Figure 2. Characterizations of the transformation of BiS@HSA/DTX mNRs and drug release in response to laser irradiation. (A) Temperature increase in BiS@HSA/DTX mNRs suspensions at different concentrations in borate buffer (0.1M, pH=8.5) subjected to laser irradiation (808 nm, 1 W/cm2) as a function of the irradiation time. (B) Plot of temperature changes vs concentration for a BiS@HSA/DTX mNRs suspension in borate buffer subjected to laser irradiation (808 nm, 1 W/cm2, 10 min). (C) Thermal NIR images of BiS@HSA/DTX mNRs suspensions at different concentrations 25 ACS Paragon Plus Environment


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subjected to laser irradiation (808 nm, 1 W/cm2, 10 min). (D) Temperature increase in 400 g/mL BiS@HSA/DTX mNRs suspensions in borate buffer (0.1M, pH=8.5) subjected to laser irradiation (808 nm) with different power densities as a function of the irradiation time. (E) Plot of temperature change vs power density of the employed laser (808 nm, 10 min). (F) Thermal NIR images of 400 g/mL BiS@HSA/DTX mNRs suspensions subjected to laser irradiation with different power densities (808 nm, 10 min). (G) Schematic illustration of the disassembly of BiS@HSA/DTX mNRs and DTX release upon laser irradiation. (H) Temperature increase and cooling curve for a 400 g/mL BiS@HSA/DTX mNRs suspension subjected to an ON/OFF cycle of laser irradiation (808 nm, 1 W/cm2). (I) TEM images and schematic pictures (insets) of BiS@HSA/DTX mNRs before (left) and after (right) laser exposure (808 nm, 1 W/cm2, 10 min). (J) DTX release profiles from BiS@HSA/DTX mNRs with/without laser irradiation (808 nm, 1 W/cm2, 10 min) at 37 oC and 50 oC, respectively.


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Figure 3. In vitro evaluation of the combinatorial therapeutic efficacy of BiS@HSA/DTX mNRs. MDA-MB-231 breast cancer cells were incubated with BiS@HSA NRs or BiS@HSA/DTX mNRs at various equivalent Bi doses. Control cells were treated with PBS. After 24 h of nanoparticle treatment, cell groups were divided in two sub-groups and subjected or not, respectively, to laser irradiation (808 nm, 0.5 W/cm2, 10 min). (A) Cell viability was quantitatively assessed by the CCK-8 assay. Cells treated with BiS@HSA/DTX mNRs and subjected to irradiation exhibited the greatest decrease in viability among the treatment groups. (B) The effects of the various treatments on cell viability was also qualitatively assessed through confocal fluorescence by co-staining the cells with propidium iodide (PI) and Calcine AM. Scale bar=100 μm. (C) Flow cytometry analysis of MDA-MB-231 cells subjected to various treatments. The cells were incubated with BiS@HSA NRs or BiS@HSA/DTX mNRs at an equivalent Bi dose of 100 μg/mL, co-stained with PI and Calcine AM and analyzed by flow cytometry.



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Figure 4. Biodistribution of BiS@HSA/DTX mNRs in nude mice bearing a MDA-MB-231 breast cancer. BiS@HSA/DTX mNRs were intravenously (i.v.) injected into nude mice bearing a MDAMB-231 breast cancer and the accumulation in the tumor and major organs was assessed by PA, fluorescence and CT imaging. (A) PA images of tumors in mice irradiated (top row) or not (bottom row) with a laser source (808 nm, 1W/cm2, 10 min) 12 h post-injection (p.i.). (B) Average PA intensity arising from the tumor area over 48 h p.i. (C) Enlarged orthogonal PA imaging views of selected tumor areas (red dotted squares in A). (D) Accumulation of BiS@HSA/DXT mNRs in major organs 48 h p.i. calculated by the quantitation of Bi by means of ICP-MS analysis. (E) CT images acquired before NMs injection and 15 min p.i. at various tilt angles (1, heart; 2, postcaval vein; 3, venae subclavia; 4, vena jugularis interna; 5, vena epigastrica; 6, kidney; 7, tumor microvessels). (F) Fluorescence images of tumor-bearing mice recorded at different p.i. times and with (top row) or without (bottom row) laser 28 ACS Paragon Plus Environment

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irradiation (808 nm, 1W/cm2, 10 min). G, Mean intensity of the fluorescence signals arising from the tumor area recorded at different p.i. times and with (red bars) or without (blue bars) laser irradiation (808 nm, 1W/cm2, 10 min).



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Figure 5. Therapeutic efficacy of BiS@HSA/DTX mNRs in nude mice bearing a MDA-MB-231 breast cancer. Mice were divided into four groups. Mice of group I received a single i.v. injection of PBS (control); mice of group II were treated with a single i.v. injection of BiS@HSA iNRs and were irradiated with laser 4 h post-injection (p.i.); mice of group III received 3 i.v. injections of BiS@HSA/DTX mNRs (DTX/body weight = 2.5 mg/kg) every 3 days; mice of group IV received 3 i.v. injections of BiS@HSA/DTX mNRs (DTX/body weight = 2.5 mg/kg) and were laser irradiated 4 30 ACS Paragon Plus Environment

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h after the first injection. Laser irradiation had a wavelength of 808 nm, a power density of 1 W/cm2 and a duration of 10 min. (A) Thermal infrared (IR) imaging of mice of the four treatment groups. (B) Tumor temperature increase in mice of the four treatment groups as a function of time. (C) Tumor growth inhibition in mice of the four treatment groups. The arrows indicated the time of injection of BiS@HSA/DTX mNRs in mice. (D) Photographs of the collected tumors. (E) Tumor inhibition efficiencies in mice of the four treatment groups. (F) MDA-MB-231 tumors’ weights at the end of treatments. (G) Body weight changes in mice of the four treatment groups. (H) H&E staining of tumor tissues harvested from mice of the four treatment groups 7 days post treatment. Scale bar = 100 μm. (I) H&E staining images of the major organs from mice of group I (top row) and group IV (bottom row). Scale bar = 50 μm.



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DTX Multiple Nanorods for

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