CT imaging-guided multimodal therapy of cancer

Accepted Manuscript PEGylated mesoporous Bi2S3 Nanostars loaded with Chlorin e6 and doxorubicin for fluorescence/CT imaging-guided multimodal therapy of Cancer

Lihong Sun, Mengmeng Hou, Lei Zhang, Dongxiang Qian, Qingquan Yang, Zhigang Xu, Yuejun Kang, Peng Xue PII: DOI: Reference:

S1549-9634(19)30006-1 https://doi.org/10.1016/j.nano.2018.12.013 NANO 1927

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Revised date:

20 December 2018

Please cite this article as: Lihong Sun, Mengmeng Hou, Lei Zhang, Dongxiang Qian, Qingquan Yang, Zhigang Xu, Yuejun Kang, Peng Xue , PEGylated mesoporous Bi2S3 Nanostars loaded with Chlorin e6 and doxorubicin for fluorescence/CT imaging-guided multimodal therapy of Cancer. Nano (2019), https://doi.org/10.1016/j.nano.2018.12.013

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ACCEPTED MANUSCRIPT PEGylated Mesoporous Bi2S3 Nanostars Loaded with Chlorin e6 and Doxorubicin for Fluorescence/CT Imaging-Guided Multimodal Therapy of Cancer

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Lihong Suna, b, Mengmeng Houa, b, Lei Zhangc, Dongxiang Qiand, Qingquan Yange,

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest

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a

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Zhigang Xua, b, Yuejun Kanga, b*, Peng Xuea, b*

University), Ministry of Education, Faculty of Materials and Energy, Southwest

Chongqing Engineering Research Center for Micro-

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b

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University, Chongqing 400715, China.

Nano Biomedical Materials and Devices, Chongqing 400715, China. Institute of Sericulture and System Biology, Southwest University, Chongqing 400716,

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c

d

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China.

The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150,

The Second Affiliated Hospital of Shenyang Medical College, Shenyang 110002, China.

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e

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China.

Corresponding authors:

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[email protected] (P. Xue), phone: +86-23-68253792 [email protected] (Y. Kang), phone: +86-23-68254056 Abstract: 142 words Manuscript: 4996 words References: 52 references Number of figures: 8

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ACCEPTED MANUSCRIPT Funding: This study is supported by Technology Innovation and Application Demonstration Grant of Chongqing (cstc2018jscx-msybX0078), financial support from Fundamental Research Funds for Central Universities (XDJK2017C001, XDJK2016A010), and National Natural

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Science Foundation of China (51703186, 31671037).

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Conflict of interest:

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There is no conflict of interest.

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ACCEPTED MANUSCRIPT Abstract Taking advantage of the mesoporous structure of bismuth sulfide nanostars (Bi2S3 NSs), a chemotherapeutic drug of doxorubicin (DOX) and a photosensitizer of chlorin e6 (Ce6) were concurrently loaded in the PEGylated Bi2S3 NSs to formulate a multifunctional

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nanocomplex (BPDC NSs) for tumor theranostics. BPDC NSs have excellent

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photothermal conversion efficiency and a capacity of yielding reactive oxygen species

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(ROS) upon laser irradiation, and can realize on-demand drug release by either pH-

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activation or thermal induction. Accumulation of the nanodrug could be monitored in real-time by infrared thermal imaging, fluorescence imaging and computed tomography

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(CT). More importantly, the combination effects of photothermal therapy (PTT), photodynamic therapy (PDT) and chemotherapy was demonstrated to dramatically

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suppress solid tumors without recurrence in vivo. Featured by the low systemic toxicity

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and high biocompatibility, this nanoplatform could be a promising derivative of Bi2S3

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Keywords

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NSs for imaging-guided theranostics of cancer.

Bismuth (Bi) chalcogenides; Doxorubicin; Chlorin e6; Controlled drug release; Cancer theranostics.

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ACCEPTED MANUSCRIPT Background Imaging-guided therapeutics, providing precise and personalized intervention in cancer therapy, has attracted considerable interests of researchers searching for efficient strategies against malignant tumors

1, 2

. The integration of imaging capability with

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various therapeutics not only provides the exact location of tumors for localized treatment

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with minimal impact on normal tissues, but also allows real-time monitoring the

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biodistribution and physiological effects of therapeutic agents

3-9

. Although simply

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combining multiple agents may compensate the intrinsic limitations of each individual treatment modality, there could be serious issues associated with their stability,

applications

10, 11

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compatibility and potential side effects, which may seriously constrain their clinical . Therefore, development of a single agent with multifunctional

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treatment modality becomes particularly necessary for efficacious tumor theranostics.

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In the past decade, photo-triggered tumor therapy becomes popular in the 12

. Among

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development of “smart” nanoplatforms to achieve unique clinical benefits

these phototherapeutics, near-infrared (NIR) light-responsive agents provide distinct

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ability in converting light energy into local hyperthermia for photothermal therapy (PTT)

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of cancer13-15. Recently, bismuth (Bi) chalcogenides have become increasingly prevalent not only as topological insulators but also as NIR-sensitive photothermal agents for biomedical applications 16-20. As a powerful clinical diagnostics, CT is featured with high spatiotemporal resolution and non-invasiveness

21, 22

. However, traditional CT suffers

from the limitations of poor contrast in soft tissues and ionizing radiation, as well as the pharmacokinetic problems of commercial CT contrast agents

23

. Usually, CT contrast

agents with higher atomic number (Z) may contribute to sharper image contrast at an

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ACCEPTED MANUSCRIPT equivalent mass concentration

24

. Given a typical high Z-element, Bi elementary

substance and its compounds with a corresponding high X-ray attenuation coefficient are promising candidates as CT contrast agents for biomedical applications

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.Particularly,

bismuth sulfide (Bi2S3)-based nanostructures are a very appealing class of materials 26-28

. In

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because of their biocompatibility, long blood circulation and cost effectiveness

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. Nevertheless, current Bi2S3-based biomedical materials are mainly

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and drug delivery

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addition, highly porous Bi2S3 nanostructures have demonstrated their capability for PTT

based on solitary therapeutic modalities, which is usually not sufficient for efficacious

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tumor eradication.

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Photodynamic therapy (PDT), as a minimally-invasive therapeutic modality for tumor ablation, induces cellular damage in tumoral tissues by producing cytotoxic 29-32

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reactive oxygen species (ROS), e.g., singlet oxygen

. As one of the most prevalently

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used photosensitizers, chlorin e6 (Ce6) has remarkable quantum yield of singlet oxygen and NIR fluorescence emission for PDT

33, 34

. However, Ce6 molecules are usually

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unstable and prone to aggregating in an aqueous environment, resulting in a short half-

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life of circulation due to rapid degradation 35. Furthermore, Ce6 aggregates in the form of large crystals also hinder the endocytotic cellular uptake

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. Alternatively, Ce6 can also

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be delivered into cancerous tissues and tumor cells intracellularly facilitated by a variety of nanocarriers

37-39

. More interestingly, Ce6 molecules can be activated specifically

within tumor region in the presence of either exogenous or endogenous stimuli, significantly reducing adverse systemic side effects 40, 41. Based on the unique characteristics of these materials above, a class of PEGylated Bi2S3 nanostars (NSs) were designed and synthesized in this study, aiming to coordinate

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ACCEPTED MANUSCRIPT both diagnostic and therapeutic functionalities by encapsulating a chemotherapeutic drug of doxorubicin (DOX) and a photosensitizer of chlorin e6 (Ce6) (Figure 1). Basically, the Bi2S3 NSs were derived from Bi2O3 nanospheres as a precursor and template after a typical hydrothermal process. Then, the surface of Bi2S3 NSs were functionalized with

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polyethylene glycol (PEG) to improve the biocompatibility and physiological stability of

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the drug carrier. The resultant Bi2S3@PEG NSs exhibited a highly porous structure for

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loading DOX and Ce6 to form Bi2S3@PEG/DOX/Ce6 (BPDC) NSs. After intravenous injection, as-synthesized nanodrugs accumulated in tumor region because of the

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enhanced permeability and retention (EPR) effect. Subsequently, DOX release was

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accelerated in more acidic tumor microenvironment. 808 nm NIR light is the mostly used for inducing local hyperthermia mediated by photothermal agents, owing to its good

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tissue penetration depth and the least absorption by water and hemoglobin13,

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.

rendering PDT

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Meanwhile, Ce6 can be activated by a 660 nm laser to produce singlet oxygen (1O2) for . By combining dual laser irradiation (808 nm and 660 nm), PTT-

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induced hyperthermia and PDT-induced ROS generation worked concurrently for

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ablation of tumorous tissue. Meanwhile, DOX release was further promoted under elevated local temperature. Furthermore, BPDC NSs were able to couple fluorescence/CT

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dual-modal imaging for cancer diagnosis, providing more precise information of the lesion and thus guiding the therapeutics.

Methods Preparation of BPDC NSs

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ACCEPTED MANUSCRIPT The BPDC NSs were synthesized following a one-step nanoprecipitation reaction method. Firstly, 10 mg of Bi2S3@PEG NSs were dispersed into 18 mL of 1×PBS (pH = 7.4). Afterwards, 10 mg of Ce6 and 2 mL of DOX·HCl (5 mg·mL-1) were added into the above dispersion under stirring, followed by introducing 80 µL of NaOH solution (1

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mol·L-1). After further stirring for 24 h at room temperature in darkness, precipitates

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obtained through centrifugation were washed with DI water to remove free DOX and Ce6,

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and BPDC NSs were finally synthesized. To calculate the loading content and encapsulation efficiency of DOX and Ce6, 0.5 mg of BPDC NSs were dissolved in 10

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mL DMSO under sonication. After centrifugation, the supernatant was collected to

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determine the amount of encapsulated DOX and Ce6 using the corresponding standard calibration curve measured from their fluorescence spectra. To assess the DOX release in

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vitro, 2 mg of BPDC NSs dispersed in 1 mL of 1×PBS was introduced into a dialysis bag,

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which was further submerged into 80 mL of saline buffer (pH=7.4 or 5.0) as the releasing medium at 37oC. At predesignated time points, 1 mL of releasing medium was withdrawn

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from the system and replaced by fresh saline buffer of the same volume. Then, the DOX

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and Ce6 release kinetics was determined using a calibration curve based on fluorescence

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spectrophotometry (Ex/Em: 485/535 nm for DOX, Ex/Em: 405/660 nm for Ce6).

Results

Synthesis and characterizations of BPDC NSs The procedures of fabricating BPDC NSs is illustrated in Figure 1. As revealed by the SEM and TEM imaging, the Bi2O3 NPs exhibited a spherical mesoporous structure, and their hydrodynamic size was measured as ~186.2 nm using DLS (Figure S1, S2).

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ACCEPTED MANUSCRIPT Originated from Bi2O3 NPs, Bi2S3 nanostructure showed a distinct star-like morphology with a hydrodynamic diameter of ~257.8 nm (Figure 2a, b). The TEM image of BPDCs with DOX and Ce6 encapsulation exhibited a well-dispersed star-like nanostructure with a hydrodynamic size of 272.8 nm (Figure S3). For a comparative study, the

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hydrodynamic size and polydispersity index (PDI) of all the intermediate products and

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BPDC NSs measured by DLS were listed in Table S1. The zeta potentials of Bi2S3@PEG

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NSs and BPDC NSs were measured as -29.7 mV and -14.1 mV, respectively (Figure 2c), and the negative surface charge may benefit their long circulation half-lives in blood

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.

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Furthermore, the elemental composition and chemical valence of Bi2O3 NPs and

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Bi2S3 NSs were investigated using X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analysis. All the diffraction peaks of Bi2O3 NPs were ascribed to a

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typical crystal form (JCPDS 898964) as shown in Figure S4a. The XPS survey spectrum

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of Bi2O3 NPs implied the co-existence of Bi and O elements, which was consistent with their chemical composition (Figure S4b-d). For Bi2S3 NSs, the full scan survey spectrum

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revealed that the sample was primarily composed of Bi, S, O and C elements (Figure 2d).

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The peak of O1s could be attributed to the absorbed oxygen species present on the particle surface, which is commonly observed in the sample exposed to the atmosphere. The peak

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representing C element was attributed to the substrate carbon film required for XPS measurement. Particularly, two strong peaks at ~157.6 and ~162.9 eV were corresponding to Bi4f7/2 and Bi4f5/2, respectively (Figure 2e)

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. The binding energy

locating at ~224.8 eV could be assigned to S2s (Figure 2f) 44. The crystallization phase of as-prepared Bi2S3 NSs was further explored by XRD. The pattern of Bi2S3 NSs showed a typical amorphous phase and all the diffraction peaks could be completely indexed to

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ACCEPTED MANUSCRIPT orthorhombic Bi2S3 (JCPDS 898964), indicating the high purity of Bi2S3 NSs (Figure 2g). Moreover, the porosity of Bi2S3 NPs was calculated based on nitrogen adsorption– desorption isotherms using Brunauer–Emmett–Teller (BET) analysis (Figure 2h, i). The BET surface area and total pore volume of Bi2S3 NSs were calculated as 28.493 m2·g−1

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and 0.149 cm3·g−1, respectively. Furthermore, the pore size distribution of Bi2S3 NSs

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indicated an average pore size of 6 nm.

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The UV-vis-NIR absorbance spectrum of Bi2S3 NSs, DOX, Ce6 and BPDC NSs

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were investigated in the range of 300-900 nm (Figure 3a). Strong optical absorbance of BPDC NSs was observed in the near infrared range of 700-900 nm, indicating a

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prominent potential as a PTT agent. Although there was a strong and wide absorbance

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peak attributed to the Bi2S3@PEG component, the characteristic peaks at 488 nm and 400/660 nm corresponding to DOX and Ce6, respectively, were still discernible from the

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absorbance spectrum of BPDC NSs particularly under higher concentrations (Figure 3b),

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implying the successful encapsulation of these two therapeutic molecules. Meanwhile, the absorbance intensity of BPDC NSs increased proportionally with NS concentration,

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suggesting their good dispersity in aqueous condition (Figure 3b, S5a). Furthermore, no

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obvious aggregation of BPDC NSs was observed in the dispersions in DI water, 1×PBS or DMEM (with 10% FBS), showing good aqueous stability (Figure S5b). Next, the hydrodynamic size of BPDC NSs dispersed in above media was continuously monitored for two weeks. There was no considerable size change or observable aggregation of BPDC NSs in any of these three solution systems over 14 days (Figure S6). Therefore, these results indicated the long-term stability of BPDC NSs in aqueous conditions. The encapsulation efficiency (EE) for DOX and Ce6 were calculated as 15.3% and 9.5%, and

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ACCEPTED MANUSCRIPT the drug loading content (DLC) for DOX and Ce6 were determined as 13.3% and 8.7%, respectively.

Photothermal effect of BPDC NSs in vitro

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The photothermal response of BPDC NSs was evaluated by exposing sample-laden

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quartz cuvettes to a NIR laser (808 nm, 2 W·cm-2). Compared to DI water, remarkable

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temperature elevation over time was observed in the dispersions of both Bi2S3 NSs and

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BPDC NSs under laser irradiation, suggesting a strong photo-induced hyperthermia effect (Figure 3c). Additionally, the temperature increase of BPDC NP suspension exhibited a

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typical concentration- and irradiation power-dependent profile (Figure 3d, e, g and Figure

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S7). The photothermal response of BPDC NS dispersions was also monitored in real-time by infrared imaging (Figure 3g), which revealed similar results as recorded by digital

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thermometer. To assess the photothermal stability of BPDC NSs, the dispersions were

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subject to periodic laser irradiation and cooling process for four cycles (Figure 3f)45. In

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vitro drug release results are provided in Supplementary data (Figure S8).

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Cellular uptake of BPDC NSs Intracellular internalization of BPDC NSs was investigated using HeLa cells (a human cervical cancer cell line) and 4T1 cells (a murine mammary carcinoma cell line). Confocal laser scanning microscopy (CLSM) was used to visualize the cellular uptake of NPs. As shown in the confocal images, the fluorescence signals of both Ce6 and DOX intensified over the time of incubation, indicating effective internalization of BPDC NSs by HeLa cells (Figure 4a, S9). Particularly, Ce6 signal was mainly located in the

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ACCEPTED MANUSCRIPT cytoplasm, while DOX signal was both observed in the nuclei and cytoplasm. In addition, flow cytometry analysis was applied to quantify the cellular uptake efficiency of BPDC NSs. The fluorescence intensity of single cell emission measured in flow analysis was corresponding to the amount of NSs internalized in each cell. The peak and mean

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fluorescence intensity shifted to higher levels after prolonged incubation time (Figure 4b,

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c), suggesting the increased cellular uptake of NSs by HeLa cells. Specifically, the

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percentage of cells taking in detectable amount of NSs increased from 38.4% in 0.5 h to 97.4% in 6 h. Similar results were also found for 4T1 cell line (Figure S10, S11). Results

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PTT/PDT-induced cytotoxicity in vitro

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of intracellular ROS generation are provided in Supplementary data (Figure S12, 13).

BPC NSs and some intermediate products were examined to investigate the PTT/PDT-

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induced toxicity on tumor cells (Figure S14). Obviously, strong orange fluorescence of

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JC-1 aggregates was observed in the group of cells incubated with BPC NSs without laser irradiation, similar to the control groups treated with only PBS or laser exposure and

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corresponding to the normal state of mitochondria without MMP depolarization.

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Meanwhile, very weak green fluorescence denoting JC-1 monomers appeared in the cells treated with BP NSs or Ce6 under laser irradiation, indicating the occurrence of mitochondrial damage due to individual PTT or PDT, respectively. In contrast, strong green fluorescence of JC-1 monomers was observed in the cells incubated with BPC NSs under laser irradiation, implying a severe level of mitochondrial damage. A similar effect mediated by BPC NPs was also observed in 4T1 tumor cells in vitro (Figure S15).

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ACCEPTED MANUSCRIPT To further assess the PTT/PDT effect mediated by BPC NSs, LIVE/DEAD cell viability assays were conducted on HeLa cells after various treatments (Figure 6a). No observable cell death was found in the cells treated solely with BPC NSs or laser irradiation compared to the blank control. However, cell death became pronounced when

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the cells were subject to Bi2S3@PEG NSs or Ce6 under laser irradiation as evidenced by

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the red fluorescence inside the irradiation spot, suggesting strong PTT- or PDT-induced

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damage of tumor cells, respectively. More importantly, the highly intensive red fluorescence of the cells treated with BPC NSs under laser irradiation indicated the most

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severe cell destruction ascribed to a combined PTT/PDT effect. To evaluate the

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biocompatibility of the nanocarrier in vitro, the cytotoxicity to somatic cells, including human umbilical vein endothelial cells (HUVECs) and L929 mouse fibroblasts (L929s),

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was studied in a wide range of concentrations using MTT cell viability assays (Figure 6b,

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S16). The cell viability was maintained at above 80% even when the cells were incubated with Bi2S3@PEG NSs at a concentration as high as 200 µg·mL-1, suggesting acceptable

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biocompatibility of the carrier material.

In vitro cytotoxicity

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The combined PTT/PDT/chemotherapy effect mediated by BPDC NSs was investigated on HeLa cells after various treatments using MTT assays. In the non-irradiated group, negligible cell death was found in those treated with Bi2S3@PEG NSs or free Ce6 in 12 h of incubation (Figure 6c). In contrast, free DOX or BPDC NSs resulted in a notable reduction of HeLa cell viability due to the chemotherapeutic effect of DOX, particularly when the drug concentration was higher than 1 g·mL-1. After applying laser irradiation,

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ACCEPTED MANUSCRIPT the cytotoxicity of Bi2S3@PEG NSs and Ce6 under higher drug concentrations was induced by PTT and PDT effects, respectively (Figure 6d). Meanwhile, the most significant damage occurred in the cells subject to combination PTT/PDT/chemotherapy mediated by BPDC NS, under which the cell viability decreased dramatically to 6.26 ±

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0.21% at an equivalent DOX concentration of 5 µg·mL-1. For the other tumor cell line we

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investigated, 4T1 cells showed similar responses to the applied recipes of drugs with

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various dosages (Figure S17). Furthermore, we discovered that the photo-induced

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both 808 nm and 660 nm lasers (Table S2, S3).

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cytotoxicity of BPDC NSs was also positively correlated to the laser power densities of

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Potential of BPDC NSs as a CT contrast agent

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The CT contrast of BPDC NSs was evaluated by acquiring the phantom images of aqueous NS dispersion at various concentrations in vitro (Figure 7a, b). It was noted that

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brightness of the CT phantom images increased linearly with the concentration of BPDC NSs, indicating the positive correlation between the CT signal intensity and nanodrug

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concentration. As a high-Z (high atomic number) element with larger X-ray interaction

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cross section, bismuth and its analogues are a very promising type of contrast agent for enhanced CT imaging 46, 47.

Pharmacokinetics and biodistribution Effective accumulation of NPs in tumorous tissue through enhanced permeability and retention (EPR) effect is a prerequisite for combination cancer therapy. Therefore, the

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ACCEPTED MANUSCRIPT pharmacokinetics of BPDC NSs in KM mice was firstly investigated by examining the blood circulation of DOX through fluorescence spectrometry. At 24 h post-injection, ~10.63 ID·g-1 (equivalent DOX dose per gram of tissue) of BPDC NPs was found in blood circulation, which was considerably higher than ~4.12 ID·g-1 of free DOX (Figure

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7c). The biodistribution of BPDC NSs in 4T1 tumor bearing Balb/c mice was similarly

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assessed by examining the content of DOX in various organs and tumors. At 24 h post-

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injection, the mice were euthanized, and the tumors as well as major organs were excised for analysis of drug biodistribution (Figure 7d, e). The BPDC NSs exhibited a notable

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uptaking rate of ~8.14 ID·g-1 in excised tumors at 24 h, which was considerably higher

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than ~1.8% ID·g-1 for free DOX.

The fluorescence imaging capacity of BPDC NSs was investigated in 4T1 tumor

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bearing BALB/c mice intravenously injected with Bi2S3 NSs, Ce6 and BPDC NSs

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(Figure 7f, S18). For the group treated with BPDC NSs, strong fluorescence was

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observed from the solid tumor region at 24 h post-injection (Figure 7f). However, for the mice treated with free Ce6, much weaker fluorescence was observed in the solid tumor

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region while much stronger fluorescence was detected in the liver (Figure S18), attributed

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to the rapid clearance of free small molecule drugs and a typical uptake by the reticuloendothelial system (RES). Distinct from the groups treated with Ce6 or BPDC NSs, only very weak background fluorescence could be observed in the group treated with Bi2Se3@PEG NSs. Collectively, these results suggested the tumor-specific accumulation and retention of BPDC NSs under the EPR effect and prolonged blood circulation.

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ACCEPTED MANUSCRIPT In vivo antitumor efficiency and biosafety of BPDC NSs When tumor volume reached ~200 mm3, all the mice were randomly divided into six groups subject to various treatments. Firstly, the temperature profiles of the mouse body shell at 24 h post-injection were recorded using an infrared thermal camera during

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NIR laser irradiation on the tumor sites (Figure 8a, S19). As compared to control groups, the temperature of tumor sites treated with Bi2S3@PEG NSs and BPDC NSs rapidly

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increased to 55.9°C and 56.1°C within 5 min of NIR laser irradiation, respectively,

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indicating a remarkable photothermal effect. Furthermore, we explored the antitumor effect of BPDC NSs in vivo by monitoring the tumor volume variation in each group

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within 14 days after various treatments (Figure 8c). Similar to the saline group, groups of

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(3), (4) and (5) indicated insufficient levels of tumor suppression under the individual effects of PTT, chemotherapy and PDT, respectively. However, laser-activated BPDC

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NSs in group (6) suppressed the growth of 4T1 tumor evidently with an impressive

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inhibition rate of 93.29% without recurrence. On the other hand, non-activated BPDC NSs could not produce a satisfactory tumor suppression effect in group (2) with an

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inhibition rate of merely 20.9%, indicating that laser irradiation was an imperative switch

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to achieve optimum therapeutic outcome. At day 14 post-treatment, all the mice were euthanized and the tumors were excised and weighted, as shown in Figure 8b. The average weight of tumors in group (6) was the lowest among all groups (Figure 8d), consistent with the terminal tumor volume measured in vivo (Figure 8c). Moreover, there was no obvious loss of body weight in all groups, suggesting a minimum systemic toxicity of BPDC NSs (Figure 8e). Finally, we examined the excised tumors in various groups histologically by hematoxylin and eosin (H&E) staining and terminal

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ACCEPTED MANUSCRIPT deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. H&E staining indicated that most tumor cells retained their regular morphology with intact nuclei in group (1) treated by saline. Compared to other control groups of (2)-(5), severe pyknosis, karyorrhexis and karyolysis of nuclei were observed in the tumoral tissues treated with

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laser-activated BPDC NSs in group (6), indicating predominant tumor cell necrosis

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resulted from combination therapy (Figure 8f). Furthermore, TUNEL assays revealed

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serious tumor cell apoptosis in group (6) as evidenced by the prevalent green

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fluorescence (Figure 8g).

Potential BPDC-induced hemolytic effect was investigated on red blood cells

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(RBCs). RBCs resuspended in DI water and 1×PBS served as the positive and negative

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controls, respectively. Owing to the imbalanced osmotic pressure in DI water, RBCs tend to lyse and thus release hemoglobin into surrounding medium. However, 1×PBS provides

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an isotonic physiological environment that maintains the RBC structure integrity. As

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shown in Figure S20, the maximum hemolytic rate of the RBCs was only 1.92% in 4 h after treatment with BPDC NSs at a concentration of 200 µg·mL−1, suggesting the good

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hemocompatibility of BPDC NSs. Moreover, routine hematology examination on the

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peripheral blood was carried out for up to a week after the mice were administered with BPDC NSs. All the key blood components showed insignificant variation within the provided reference ranges of healthy mice (Figure S21, Table S1). Next, the major organs of treated mice were excised at day 14 for histopathological analysis by H&E staining (Figure S22). There was no apparent inflammation or lesion occurred at either cellular or tissue level among all treated groups.

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ACCEPTED MANUSCRIPT Discussion Excellent photothermal conversion capacity is always an essential feature of a mediator for efficacious PTT. A terminal temperature of 47.1oC could be achieved within 10 min of irradiation at the BPDC NS concentration of 60 ppm (Figure 3d), exceeding the

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threshold temperature of 43oC for tumor ablation. Moreover, there was no observable

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alternation in the peak temperature of each cycle during the repeated photothermal

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responses, confirming a good photothermal stability of BPDC NSs (Figure 3f). In another

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aspect, the acidity-promoted drug release could be attributed to the protonation of the amino groups in DOX molecules, which facilitated drug dissolution in the release 48

. Given the more acidic tumor microenvironment, as-synthesized BPDC NSs

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medium

could potentially realize effective tumor-specific DOX delivery (Figure S8). Moreover, a

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photo-sensitive drug release behaviour can be attributed to the highly responsive

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photothermal capability of Bi2S3, which weakened the carrier-drug interaction upon laser

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activation and resulted in an enhanced release rate as also reported in prior literature 49, 50. Efficient cellular uptake of therapeutic agents is critical to ensure the sufficient

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bioavailability of drugs at tumor site and thus the efficacy of treatment. Both Ce6 and

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DOX can produce fluorescence emission under specific photo-excitation, which can be utilized to trace the internalized BPDC NPs without additional fluorescence markers. Results of both confocal imaging and flow cytometry indicated a remarkable cellular uptake efficiency of BPDC NSs (Figure 4). Reactive oxygen species (ROS) play a key role in cell signalling and homeostasis, and are a facilitator for photodynamic therapy (PDT). In the present study, the production of intracellular ROS was characterized in vitro using DCFH-DA, which can be hydrolysed into DCFH intracellularly and further

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ACCEPTED MANUSCRIPT oxidized into fluorescent dichlorofluorescein (DCF) under oxidative stress. Since the fluorescence spectra of DOX and DCF overlapped, DOX-free Bi2S3@PEG/Ce6 (BPC) NSs were utilized to avoid the interference with ROS detection. Cells treated with free Ce6 were concurrently examined for comparison. The results verified that BPC provided

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a potent nanocarrier for more efficient delivery of the photosensitizer Ce6 than its free

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molecular forms (Figure 5).

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Apoptosis is closely related to the dysfunction of mitochondria, which can be

conversion process

51, 52

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induced by the oxidative stress from ROS and hyperthermia during photothermal . Mitochondrial membrane potential (MMP) was a critical

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indicator of the physiological status of mitochondria, which can be monitored using a JC-

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1 fluorescence staining kit. JC-1 is a cationic carbocyanine dye, which tends to aggregate in the matrix of normal mitochondrial and produces orange fluorescence (emission peak:

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590 nm). However, the MMP of damaged mitochondria decreases and causes conversion

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of JC-1 from aggregated state into monomers, which diffuse into cytoplasm and emit green fluorescence (emission peak: 520 nm). Both of HeLa cells and 4T1 cells presented

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severe mitochondrial damage mediated by BPC NPs, suggesting a strong combined

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PTT/PDT effect (Figure S14, S15). When evaluating the BPC NSs cytotoxicity using Live/Dead cell viability assay, the results were well in accordance with those obtained via JC-1 staining. Cytotoxicity of BPDC NSs from MTT assay quantitatively demonstrated a capable and highly responsive nanoplatform to realize the combined treatment of PTT/PDT/chemotherapy. A relatively longer circulation half-life of BPDC NSs compared to free DOX favors effective drug accumulation in tumors via the EPR effect (Figure 7c). Biodistribution of free DOX in tumor site further confirmed an enhanced drug retention

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ACCEPTED MANUSCRIPT mediated by BPDC nanocarrier (Figure 7d). Fluorescence and CT properties of BPDC NSs could potentially realize multimodal imaging for cancer diagnosis and staging for clinical

usages.

Encouraged

by

the

promising

effect

of

combined

PTT/PDT/chemotherapy mediated by BPDC NSs in vitro, we proceeded to an in vivo

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investigation using BALB/c mice bearing 4T1 tumors. The results demonstrated a

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remarkable therapeutic efficacy for ablating solid tumors under the combinatorial effect

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of local hyperthermia, intracellular ROS and chemotherapeutic toxicity mediated by BPDC NSs (Figure 8). The potential toxicity of BPDC NSs were assessed in terms of

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hemocompatibility and histocompatibility. The results provided a clear evidence that

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BPDC NPs caused minimum systemic toxicity or damage on major organs, which could

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favor the prolonged circulation in the body after intravenous administration. In summary, PEGylated mesoporous Bi2S3 NSs were synthesized and

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concurrently loaded with a photosensitizer (Ce6) and a chemotherapeutic drug (DOX) as

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a robust nanoagent for imaging-guided multimodal tumor therapy. At the in vitro level, on-demand drug release was demonstrated to be precisely regulated by the endogenous

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factor of pH condition as well as the exogenous stimuli of laser irradiation. The strong

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local hyperthermia generation and the yield of ROS mediated by BPDC NSs were simply realized by laser irradiation. After systemic administration in tumor bearing mice, BPDC NSs exhibited rapid accumulation inside the tumor as evidenced by tracing the fluorescence signal of Ce6. A remarkable antitumor efficacy was achieved with the aid of BPDC NSs upon laser irradiation, resulted from the combined PTT/PDT/chemotherapy effect. Benefited from the good biocompatibility, no obvious hepatotoxicity or systemic toxicity was observed during BPDC NS-mediated tumor therapy. This capable

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ACCEPTED MANUSCRIPT theranostic nanoagent has a good potential to coordinate multiple bioimaging modalities, including infrared thermal imaging, fluorescence imaging and high contrast CT. Therefore, this proof-of-concept work may enlighten further explorations of inorganic metal chalcogenides for applications in multimodal imaging-guided tumor combination

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therapy.

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Schematic illustration of the synthetic procedure of PEGylated mesoporous Bi2S3 nanostars encapsulating DOX and Ce6 (BPDC NSs) as a multifunctional platform

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for cancer theranostics.

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Figure 2. Physicochemical characterizations: (a) a high-resolution TEM image of Bi2S3

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NSs (scale bar: 200 nm); (b) hydrodynamic diameter of Bi2S3 NSs measured by DLS; (c) zeta potentials of intermediate and final products; (d-f) XPS survey spectra of Bi2S3 NSs,

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including (d) full scan XPS survey spectrum of Bi2S3 NSs and a core level XPS spectrum

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corresponding to (e) Bi4f and (f) S2S; (g) XRD patterns of Bi2S3 NSs; (h) N2 adsorption– desorption isotherm and (i) the corresponding pore size distribution of Bi2S3 NSs.

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Figure 3. Photothermal characterizations: (a) UV-vis-NIR absorption spectra of

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Bi2S3@PEG NSs, free DOX, free Ce6 and BPDC NSs; (b) UV-vis-NIR absorption spectra of BPDC NSs dispersed in DI water at various concentrations; (c) temperature

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variation in the dispersions of BPDC NSs (100 ppm) , BP NSs (100 ppm) and DI water

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under NIR laser irradiation for 10 min (808 nm, 2 W·cm-2); (d) temperature variation in BPDC NS dispersions at various concentrations (0 to 100 ppm) under NIR laser

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irradiation for 10 min (808 nm, 2 W·cm-2); (e) temperature variation of BPDC NS dispersion (100 ppm) under NIR laser irradiations with different power intensity for 10 min; (f) temperature variation of BPDC NS dispersion (100 ppm) under cyclic laser exposures (10 min of laser irradiation per cycle); (g) thermographic imaging of sampleladen cuvettes corresponding to (c), (d) and (e).

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ACCEPTED MANUSCRIPT Figure 4. Characterizations of cellular uptake of BPDC NSs: (a) Confocal fluorescence images of HeLa cells incubated with BPDC NSs for 1 h or 4 h. The fluorescence signals of DOX and Ce6 are displayed in green and red colors, respectively (scale bars: 20 µm). Images in 2nd and 4th rows denote the magnified areas enclosed in dashed box in 1st and

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3rd rows, respectively. (b) Flow cytometry analysis of cellular uptake of BPDC NSs in

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HeLa cells after different incubation time. (c) Mean intensity of fluorescence emission

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from the cells corresponding to (b). (d) Flow cytometry dot plots with BPDC uptaking

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rate of the cells corresponding to (b).

Figure 5. Characterization of intracellular ROS generation using DCFH-DA probe:

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confocal fluorescence images of HeLa cells subject to Ce6 or BPC NSs with or without

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660 nm laser irradiation (scale bars: 20 µm). Images in 2nd, 4th, 6th and 8th rows are the

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magnified areas enclosed in dashed box in 1st, 3rd, 5th and 7th rows, respectively. Figure 6. (a) Live/Dead cell viability assays on HeLa cells under various treatments. The

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dashed circles denote laser irradiation spots (scale bars: 200 µm, 0.232 mm2 per spot); (b) Viability of HUVECs and L929 cells after treatment with Bi2S3@PEG NSs at various

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concentrations (0 to 200 µg·mL-1); Viability of HeLa cells after treatment with

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Bi2S3@PEG NSs, free Ce6, DOX or BPDC NSs without (c) or with (d) laser irradiation. Figure 7. (a) In vitro CT images of BPDC NSs; (b) CT values of the aqueous dispersions of BPDC NSs at various concentrations corresponding to (a); (c) In vivo pharmacokinetic curves over 24 h after intravenous injection of free DOX or BPDC NSs; (d) Biodistribution of free DOX or BPDC NSs at 24 h post-injection; (e) The fluorescence image of excised organs and tumors at 24 h post-injection; (f) Fluorescence images of

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ACCEPTED MANUSCRIPT BALB/c mice bearing 4T1 tumors before and after the administration of BPDC NSs (dashed circles denote the location of solid tumor). Figure 8. Treatment of tumors in vivo with BPDC NSs: (a) infrared thermal images of tumors after intravenously injection with various agents upon NIR laser irradiation; (b)

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photographs of the excised tumors at day 14 after various treatments; (c) tumor volume variation after various treatments; (d) weight of the excised tumors at day 14 after various

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treatments; (e) change in mouse body weight after various treatments; histological

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analysis of tumor sections using (f) H&E staining (black dashed boxes denote magnified

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sections) and (g) TUNEL staining.

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ACCEPTED MANUSCRIPT Graphical abstracts

Highly biocompatible PEGylated mesoporous bismuth sulfide nanostars encapsulating

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doxorubicin and chlorin e6 (BPDC NSs) were facilely synthesized for tumor theranostics.

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BPDC NSs, as a potent contrast agent, also enable specific multimodal imaging of tumors,

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including infrared thermal imaging, fluorescence imaging and computed tomography (CT). Moreover, aided by the passive targeting effect, the accumulation of BPDC NSs

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contributed to the combination effects of photothermal therapy, photodynamic therapy

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and chemotherapy of solid tumors. Collectively, BPDC NPs may serve as a promising nanomedicine for highly efficacious tumor suppression and eradication with imaging

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guidance.

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Figure 1

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Figure 8