Rodlike MSN@Au Nanohybrid-Modified Supermolecular

Jan 22, 2019 - Department of Nuclear Medicine, Tianjin Medical University General ... Nanoparticles for Clearable FL/CT/MSOT Imaging-Guided PTT/PDT ...
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Biological and Medical Applications of Materials and Interfaces

Rod-like MSN@Au Nanohybrids modified supermolecular photosensitizer for NIRF/MSOT/CT/MR Quadmodal Imaging Guided Photothermal/Photodynamic Cancer Therapy Shan Yang, Qing You, Lifang Yang, Peishan Li, Qianglan Lu, Siyu Wang, Fengping Tan, Yanhui Ji, and Nan Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19565 • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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Rod-like MSN@Au Nanohybrids modified supermolecular photosensitizer for NIRF/MSOT/CT/MR Quadmodal Imaging Guided Photothermal/Photodynamic Cancer Therapy Shan Yanga, Qing Youa, Lifang Yanga, Peishan Lia, Qianglan Lua, Siyu Wanga, Fengping Tana, Yanhui Ji* and Nan Li* a

Tianjin Key Laboratory of Drug Delivery & High-Efficiency, School of

Pharmaceutical Science and Technology, Tianjin University, 300072 Tianjin, PR China. * Co-corresponding author: Department of Nuclear Medicine, Tianjin Medical University General Hospital, 300052 Tianjin, PR China. E-mail address:[email protected] Corresponding author: Tianjin Key Laboratory of Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, 300072, Tianjin, PR China. Tel.:+86-022-27404986. E-mail address: [email protected].

Key words: Nanorods, Photothermal/Photodynamic Therapy, Supra photosensitizers, Host-guest reaction, Quadmodal Imaging 1

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ABSTRACT Recently, rod-like nanomaterials with specific aspect ratio (AR) for efficient cellular uptake have received enormous attention. For functional nanomaterials, such as photothermal agents, large surface areas for their rod-shape exterior that increase the amount of light absorbed would lead to the higher absorption coefficient as well as drug loading property. In this project, we coated rod-like mesoporous silica with gold nanoshell (MSNR@Au hybrid), modifying with ultrasmall gadolinium (Gd) chelated supramolecular photosensitizers TPPS4 (MSNR@Au-TPPS4(Gd)), which could be applied to near-infrared fluorescence (NIRF) / multispectral optoacoustic tomography (MSOT) / computed tomography (CT) / magnetic resonance (MR) imaging and imaging guided remotely controlled PTT/PDT combined antitumor therapy. Gold nanoshell, as a perfect PTT agent, was used to assemble the rod-like mesoporous silica nanoparticles with larger superficial area and higher drug loading, thus obtaining MSNR@Au hybrid. HS-β-CD, which was used as the host, was adsorbed on gold nanoshell (MSNR@Au-β-CD) to link TPPS4(Gd) through host-guest reaction, thus forming CD-TPPS4 supramolecular PSs (supraPSs). Compared with conventional PSs, supraPSs had the host screens, which could reduce the self-aggregation of the TPPS4, and consequently generate 1O2 with high-efficiency. The quad-modal imaging in vivo of MSNR@Au-TPPS4(Gd) nanoparticles revealed that an intensive tumor uptake effect after injection. In vivo antitumor efficacy further testified that the synergistic therapy, which was more efficient than any other mono-therapy, exhibited an excellent tumor inhibition therapeutic effect. As a result, it’s encouraged to further 2

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explore multifunctional theranostic nanoparticles based of gold shells for cancer combination therapy.

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1.INTRODUCTION Over the past years, the utilization of rod-like nano-materials in drug delivery has been receiving significant interests in cancer therapy. Due to the specific aspect ratio (AR), it has shown a disproportionate impact on biological outcome1-2, including particle transportation3-4, biodistribution5, and biocompatibility6-7. Among them, gold nanoparticles with rod-like shape are widely applied in potential biomedical applications8-11. Owing to the unique surface plasmon resonance (SPR), gold nanoparticles have been a widely employed photothermal therapy agents in the treatment of cancer12-13. Relatively, Au nanorods (AuNRs) possess tunable aspect ratios, superior spectral bandwidth and more effective photothermal energy conversion than gold nanosphere14-16. However, AuNRs have limitations in some aspects such as smaller superficial area and lower drug loading. To address these problems, some rod-like nanoparticles combined silica and gold rise in response to the proper time and conditions, for instance, Au shell coated rod-like mesoporous silica nanoparticles17-18, gold nanorods-silica Janus nanoparticles19, gold nanorod cores coated hollow silica nanoparticles (Au@HSNs)20, rare-earth-gold core–shell nanorods (RE-Au)21 and so on. Among these types of nanoparticles, gold shell coated rod-like mesoporous silica nanoparticles (MSNR@Au shell) exhibited excellent advantages, such as larger superficial area, higher drug loading, as well as better photothermal therapy (PTT) effect. Photothermal therapy (PTT) utilizes photoabsorbing agents (PTA) to produce hyperthermia from laser energy to cause cancer cell death. However, it’s hard to 4

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completely eliminate tumors by PTT alone with the least side effects, even if the nanomaterial usage is by optimization as far as possible. Photodynamic therapy (PDT), which provides toxicities to cancer cells by reactive oxygen species (ROS) for instance singlet oxygen (1O2) or other radical species relying on the photosensitizers (PS) transferring laser energy to the surrounding oxygen22, has been reported to combine with PTT, for the dramatically improved therapeutic efficacy and effectively decreased side effects23. Unhappily, the broadest used PSs, such as porphyrins and phthalocyanines, usually causes limited 1O2 quantum yield and low biocompatibility because of the serious aggregation because of their hydrophobic characteristics and p-p interaction

24-26.

To solve this problem, much ramification with kinds of charged

hydrophilic substituent groups have been exploited27-28. Unfortunately, the chemical synthesis of these chemical entities usually need intricate molecular designing and more time. On the other hand, the bioactivity of ionic photosensitizers is hard to be reasonably controlled because cationic PSs may give rise to serious aggregation with serum constituents while anionic PSs are hard to be taken by cells29-32. In recent years, supramolecular PSs (supraPSs), as supramolecular complexes with their favorable photodynamic properties, have garnered extensive research interests. Compared with conventional PSs, supraPSs had the host screens, which could weaken the self-aggregation of the centric PSs, and consequently guarantee the generation

of

1O

2

with

high-efficiency.

5,10,15,20-Tetrakis

(4-sulfonatophenyl)-porphyrin (TPPS4), as the most used second generation PDT sensitizers, not only can form supra molecules with cyclodextrin (CD) to enhance the 5

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PDT effect, but also can be chelated with metal ions, such as gadolinium ion, to exhibit MR imaging. In this paper, mono-(6-mercapto-6-deoxy)-beta-cyclodextrin (HS-β-CD) was utilized as the host and TPPS4 was the guest of the host-guest reaction. The cavities of cyclodextrin molecules were hydrophobic, which could provide space for conjugating with TPPS4. When TPPS4 added into cyclodextrin, it could be embedded into the cavities of cyclodextrin partly or entirely through non-covalent force to form stable inclusion complexes, which could reduce the aggregation of TPPS4 therefore enhanced PDT effect. To synthesize our final formulation, we first utilized HS-β-CD and gold shell (MSNR@Au shell) to synthesize MSNR@Au-β-CD through the Au-S bond. Then, TPPS4(Gd) was alternatively deposited onto MSNR@Au-β-CD via assembly strategy33-36 with the formation of CD-TPPS4 supraPSs. To achieve the goals of effective antitumor efficiency, it’s a key to obtain the accurate location of tumor37. In the past years, many nanomaterials were designed to be utilized for exact image formations, for example, near-infrared fluorescence (NIRF), multispectral optoacoustic tomography (MSOT), computed tomography (CT), magnetic resonance (MR) and so on. Nevertheless, to achieve ideal imaging requirements, multi-mode imaging of nanostructures is needed because single-mode imaging has some defects38. For instance, the NIRF probes have lower-resolution and weaker tissue penetration, MR imaging suffers from poor sensitivity, and it’s difficult for CT imaging to identify minor variation of soft tissues. Therefore, quadmodal imaging, which could overcome shortcomings of different imaging modals to obtain 6

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more accurate and adequate information in vivo, could be designed. In our work, a multifunctional all-in-one platform with quadmodal imaging and combined antitumor efficiency was structured to achieve more accurate and efficient therapy. In this study, we creatively fabricate gadolinium (Gd) chelated TPPS4 functionalized MSNR@Au nanohybrids (MSNR@Au-TPPS4(Gd)) through the Au-S bond using HS-β-CD as a host-guest linker. To our knowledge, this combination has never been reported before. The prepared MSNR@Au-TPPS4 (Gd) nanoparticles could achieve an NIRF/MSOT/CT/MR imaging guided PTT/PDT combined anti-tumor

therapy

with

a

number

of

outstanding

features

(Fig.1).

(1)

MSNR@Au-TPPS4 (Gd) nanoparticles with an AR of about 2 are endocytosed with priority by an effective uptake mechanism which can be used to distinguish the length of rods; (2) With the form of supraPSs, MSNR@Au-TPPS4 (Gd) nanoparticles can ensure the generation of 1O2 with high-efficiency as the host screens of supraPSs can reduce the self-aggregation of the centric PSs; (3) Under the near infrared (NIR) light irradiation, MSNR@Au-TPPS4 (Gd) can kill tumor cells by generating a localized hyperthermia and release TPPS4, therefore realizing combined PTT/PDT effect; (4) The quadmodal imaging by MSNR@Au-TPPS4 (Gd) provides tumor information with accuracy from the spectrum of magnetic perspective, electronic, and optical with various imaging agents in comparison with mono modal imaging. In a hopeful manner, the quadmodal imaging guided combined therapy can enhance the anticancer efficacy to a large extent.

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Figure 1. Diagrammatic drawing of the synthetic routes and theranostic process of MSNR@Au-TPPS4 (Gd) nanoparticles for multimodal NIRF/MSOT/CT/MRI imaging guided photothermal therapy and supraPSs-based photodynamic therapy.

2. MATERIALS AND METHODS 2.1. Materials. NaBH4, L-ascorbic and TPPS4 were all purchased from Heowns Biochem Technologies LLC. HS-β-CD was obtained from Shandong Binzhou Zhiyuan Biotechnology Co. Ltd acid. Gd-(NO3)3·6H2O was bought from J&K Scientific Ltd. Chloroauric acid (HAuCl4), (3-aminopropyl) triethoxysilane(APTES), dimethyl

sulfoxide

(DMSO),

4′,6-diamidino-2-phenylindoles

calcein (DAPI),

acetoxymethyl

ester

(calcein

1,3-diphenylisobenzofuran

AM),

(DPBF),

5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide (PI) were bought from Sigma-Aldrich. 2.2. Preparation of MSNR@Au-TPPS4 (Gd) Nanoparticles. 2.2.1. Synthesis of MSNR Rod-like mesoporous silica nanoparticles (MSNR) were obtained via a modified 8

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procedure according to the literatures17-19. After washed with methanol, the products were re-dispersed in acidic methanol (5 ml of concentrated hydrochloric acid in 50 ml methanol) and heated to 60 °C, followed by refluxing under magnetic stirring to get rid of CTAB. After that, mesoporous silica was obtained by centrifugation. Then, they were re-dispersed in the mixture of ethanol and (3-aminopropyl) triethoxysilane (APTES) and heated to 80°C-90°C, followed by refluxing for 5-6 h under stirring to form MSNR-NH2. Finally, mesoporous silica was centrifuged, washed with ethanol for the first time and with water for another two times, finally stored into 20 ml of double distilled water for further usage. 2.2.2. Synthesis of MSNR@Au shells. Seed-induced growth method38-39 was utilized to synthesis the MSNR@Au shells. Briefly, 0.1 M of HAuCl4 (100 μL) was mixed with double distilled water (30 mL) at first; Second, the colorless transparent solution was obtained when the pH value was adjusted to more than 9.0 by adding NaOH solution (0.02 M). After that, MSNR-NH2 (1.8 mL) solution was put in the colorless transparent solution above at once and kept stirring for 30 min. Finally, HAuCl4 was reduced by adding 1.2 mL of NaBH4 solution (0.01 M) dropwise. The obtained claret-colored solution (MSNR@Au seeds) was separated by centrifugation after stirring for another 6 h, and finally re-dispersed in 4 mL of double distilled water. To obtain the MSNR@Au shells, 150 μ L of HAuCl4 (0.1 M) was mixed with 40 mL of potassium carbonate (K2CO3) solution and stirring for at least 40 min till the color of the solution changed from yellow into colorless, followed by adding 250 μ L of MSNR@Au seeds solution above and 9

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L-ascorbic acid solution successively. It’s indicated that the Au nanoshells had formed successfully when the color of the solution changed into light blue-purple. The MSNR@Au shells nanocomposites were obtained by centrifugation Finally, the MSNR@Au shells were re-dispersed in 1 mL of double distilled water. 2.2.3. Preparation of MSNR@Au-β-CD. MSNR@Au- β -CD were prepared by using the bond reaction between Au and S40. First, the HS-β-CD (2 × 10-5 M) and 1 mL of the MSNR@Au shells solution obtained before were mixed, followed by gently stirred at 25 ℃ overnight. Finally, MSNR@Au- β -CD was centrifuged, washed with water, and stored after vacuum drying for the usage of next step. 2.2.4. Preparation of MSNR@Au-TPPS4(Gd) Firstly, according to the literatures41-42, TPPS4(Gd) was prepared by simply incubating TPPS4 in a Gd(NO3)3 aqueous solution overnight. After that, the obtained MSNR@Au-β-CD was put into 3 mL of TPPS4(Gd) solution. The mixture was stirred for 15 min, thus leading to the adsorption between TPPS4(Gd) and MSNR@Au-β-CD via host-guest interaction. Finally, MSNR@Au-TPPS4(Gd) was centrifuged and washed with water. In TPPS4 loading experiments, various concentrations of TPPS4 (Gd) solution were added into the same of MSNR@Au-β-CD PBS solutions (pH 7.4) for 15 min under gentle stirring. The acquisitions were centrifuged and washed with PBS. For exploring the loading ratio of TPPS4 (Gd), the washing liquor were collected in full to measure the absorbency at 414 nm. The loading ratio of TPPS4 was showed as 10

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Loading ratio = ( MTPPS4(Gd) - MuTPPS4(Gd) ) / MMSNR@Au-β-CD × 100% where MTPPS4(Gd), MuTPPS4(Gd), and MMSNR@Au-β-CD are the amount of TPPS4 (Gd), unloaded TPPS4 (Gd), and MSNR@Au-β-CD, respectively. The instruments of characterizations are all listed in the supporting information. 2.3. In Vitro MSOT/CT/MR Imaging. In vitro MSOT imaging, the MSNR@Au-TPPS4(Gd) nanoparticles were conducted by a MSOT imaging system. The MSOT signals of different concentrations of MSNR@Au-TPPS4(Gd) solutions, corresponded to different Au concentrations, were measured by a high resolution photoacoustic tomography system at the wavelength of 808 nm. In the MSOT system reported here, a tunable optical parametric oscillator laser delivered pulses with the wavelengths ranging from 680 nm to 1064 nm. For tomographic recording, a sphere-focused ultrasound transducer with high center frequency was mounted on a rotation stage. The detected photoacoustic signals were digitized by a data acquisition (DAQ) card and finally stored by the computer controlling system. In vitro CT imaging, different formulations of MSNR@Au-TPPS4(Gd) nanoparticles with various Au concentrations (0, 17.01, 34.02, 68.05, 102.07, and 204.15 μg/mL) were added into tubes and performed by small mice X-ray CT, Here are the relative parameters: Voltage: 90kV,Current: 180μA,field of view (FOV): 73mm, Scan Time: 4.5min. In vitro MRI studies, MSNR@Au-TPPS4(Gd) nanoparticles with different Gd3+ concentrations were tested by a 3.0 T MR instrument. Parameters are listed in the 11

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supporting information in details. 2.4. In vitro Photothermal Performance. MSNR@Au-TPPS4(Gd) solutions (0.5 mL) with various concentrations and different samples (0.5 mL) (H2O, free TPPS4, MSNR@Au shell, and MSNR@Au-TPPS4(Gd)) were irradiated with 808 nm laser (1.5 W/cm2). Digital thermometer was used to measure the solution temperature changes at predesigned time intervals, and infrared thermal camera was utilized to capture the thermal pictures of different formulations. In order to assess the photothermal conversion efficiency (η), continuous 808 nm laser irradiations were used to measure the temperature variation of MSNR@Au-TPPS4(Gd) (containing 163 μg/mL of MSNR@Au- β -CD and 35.4 μg/mL of TPPS4) solution for 300s followed by a cooling stage. For photothermal cycling tests, 0.5 mL of MSNR@Au-TPPS4(Gd) solution was treated with four on−off 808 nm laser irradiations (1.5 W/cm2). 2.5. In Vitro Photodynamic Performance. DPBF was used as the 1O2 sensor to assess the generation of 1O2. 20 μL of DPBF acetonitrile solution was added into 2ml of TPPS4 (35.4 μg/mL), MSNR@Au-CD (163

μg/mL),

and

MSNR@Au-TPPS4(Gd)

(containing

163

μg/mL

of

MSNR@Au-CD and 35.4 μg/mL of TPPS4) respectively. After laser irradiation (660 nm, 1W/cm2), the absorbency at 410 nm of DPBF in the various formulations was recorded at appointed time points. 2.6. Cellular experiments. For visualizing the cellular uptake of different formulations, 4T1 cancer cells (5 12

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× 105 cells/well) were cultivated with free TPPS4 or MSNR@Au-TPPS4(Gd) (35.4 μg/mL of TPPS4) for 4 h. Afterwards, the cells above were washed using PBS solution, dyed with DAPI, and conducted by a CLSM. The ROS in the cell generated through MSNR@Au-TPPS4(Gd) and TPPS4 was assessed by using DCFH-DA. 4T1 cells were first cultured with different formulations (saline, free TPPS4, and MSNR@Au-TPPS4(Gd)) for 12 h. Afterwards, the cells above were washed using PBS and cultivated with 10 μM of DCFH-DA. Then they were washed with PBS again, followed by irradiated with 660 nm laser (1 W/cm2). DCFH could be of oxidization to dichlorofluorescein (DCF) in cytoplasm in the presence of ROS43, which was a highly fluorescent derivative. The results could be measured by CLSM. The cytotoxicity of different formulations was testified by MTT assay. First, the 4T1 cells, 5 × 104 per well in 96-well plate, were cultured in the dark with different concentrations of different formulations for 24h. For phototoxicity of the formulations, the cells were incubated for 6h, and then treated with NIR laser irradiation, followed by a further cultivation for 18 h. Afterwards, another 4 h of cultivation was needed after MTT solution was injected into each well. Finally, the formazancrystals was dissolved by adding 500 μL of DMSO after the culture media were separated. Microplate reader was made use of testing the absorbance at 490 nm. Live/dead cell costaining studies were utilized to further evaluate the tumor cell-killing ability. After being pre-seeded into CLSM dishes, 4T1 cells (1 × 106) were cultivated with different formulations (PBS, 35.4 μg/mL of TPPS4, 163 μg/mL of 13

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MSNR@Au-CD and 200 μg/mL of MSNR@Au-TPPS4 (Gd)) for 12 h, and followed by treating with or without the NIR laser irradiation. Then, the calcein-AM and PI were added in an orderly way for a further cultivation after washing the cells. At last, the cells images were obtained by a CLSM after being washed several times. 2.7. Hemolysis experiment. First, the samples of blood were acquired from volunteers, followed by centrifugation to separate erythrocytes from the serum. After that, the erythrocytes were of dilution to 10% with PBS solution after washed with 0.9% saline. Thereafter, 400 μL of above suspension was injected into 1.6 mL of DI water, PBS, and MSNR@Au-TPPS4(Gd) with various concentrations. Afterwards, the mixtures were incubated and then centrifuged. Eventually, UV–vis spectrometer was utilized to measure the absorbance of the supernatants. 2.8. Tumor Model. All of the BALB/C mice and nude mice were obtained from HFK (Beijing) Bioscience Co., Ltd. The whole of animals’ laboratory procedures were carried out in adherence with the guiding principles of Tianjin University. 4T1 cancer cells (2 × 106) were subcutaneously injected into the mice to obtain the tumor models. Animal experiments were carried out once the size of tumor reached about 100 mm3. 2.9. In Vivo Quadmodal Imaging. For FL imaging in vivo, free TPPS4 or MSNR@Au-TPPS4 (Gd) (TPPS4, 1.0 mg/kg) was injected into tumor-bearing nude mice intravenously. Then, the IVIS Lumina imaging system (Caliper, USA) was used to measure the time-dependent 14

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fluorescence images at the specific wavelength (excitation wavelength: 637 nm; emission wavelength: 660-700 nm). Finally, the mice were of sacrifice, ex vivo imaging was tested by moving out the tumors and the major organs. For MSOT imaging in vivo, nude mice bearing 4T1 cells were treated with MSNR@Au-TPPS4 (Gd) nanocomposites (25 mg/kg) by intravenous injection. Thereafter, the MSOT images were obtained at pre-designed time points. (Tunable optical parametric oscillator laser: Solar, Belarus; sphere-focused ultrasound transducer: Precision Acoustics, UK) For CT imaging in vivo, the CT images were captured by a X-ray CT equipment before and after MSNR@Au-TPPS4 (Gd) nanoparticles (25 mg/kg) were injected into nude mice with tumor intratumorally. Here are the relative parameters: Voltage: 90kV, Current: 180μA,field of view (FOV): 73mm, Scan Time: 4.5min. For MR imaging in vivo, T1-weighted signals were recorded by a 3.0 T MR instrument before and after the MSNR@Au-TPPS4(Gd) nanoparticles(1.27 μ mol Gd/kg mice) were injected into tumor-bearing mice. Parameters are listed in the supporting information in details. 2.10. Antitumor Efficiency in Vivo. When the volume of tumor was about 100 mm3, the female BALB/C mice with tumors were stochastically grouped as four groups to measure the antitumor effect: (1) PBS, (2) Free TPPS4 + 660 nm; (3) MSNR@Au shell + 808nm; (4) MSNR@Au-TPPS4(Gd) + 808/660 nm. Caliper was used to measure the tumor volume. The body weights of mice were measured by an electronic balance and 15

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evaluated by an equation (volume = width2 × length/2)

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44.

In addition, the survival

rate of each group was recorded after the therapies and finally the survival curves were plotted. Last day, the organs as well as tumors were taken out, followed by dyed with hematoxylin as well as eosin (H&E) for histopathology studies. 2.11. Statistical Analysis. Herein, mean plus & minus standard deviation as well as analysis of variance were utilized to be Statistical analysis methods. 2-sample t-test was utilized to calculate the P values. Significant difference was represented by the probability level of 95%.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of MSNR@Au shell. MSNR@Au shells were structured through developing the gold shell on the mesoporous silica nanorod (MSNR). The MSNR core (Fig. 2A), synthesized by co-condensation45, showed a perfect morphology. After that, the amino was modified onto the surface of MSNR to acquire the MSNR-NH2, which was utilized to yield MSNR@Au seeds and subsequently to obtain gold nanoshells (MSNR@Au shells). On the basis of the in situ reduction method46, Au3+ ions were first absorbed on the surface of MSNR-NH2 by electrostatic incorporation, followed by reduced on site to yield gold seeds (MSNR@Au seeds). After that, the gold seeds became thicker progressively and developed into homogeneous gold nanoshells (MSNR@Au shells). Fig. 2A showed that the MSNR nanoparticles had mean length of 190 ± 13.1 nm and 16

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width of 95 ± 4.8 nm; Fig. 2B illustrated that the Au nanoparticles were attached to the outside of MSNR to form MSNR@Au seeds with a success. Gold seeds gradually grew thicker, and finally became the nanoshell-capped MSNR (MSNR@Au shells) with a rod-like morphology( Fig. 2C). Moreover, the elemental mapping analysis (Fig. 2F, G, H, I) exhibited that there were three components of Si, O and Au, all with a rod-like morphology, revealing that the MSNR@Au shell were achieved. Acidic methanol (1 ml of concentrated hydrochloric acid in 50 ml methanol) was used to obtain the mesoporous silica rod. IR spectra were utilized to test if the CTAB was removed completely (Fig. 2D). For the initially formed silica rod (the black curve), there was a strong absorption peak at 2922 cm-1 and a small absorption peak at 2850 cm-1, both of which belonged to C-H stretching vibrations. Nevertheless, there was no peak at 2922 cm-1 and 2850 cm-1 after CTAB extraction (the red curve), demonstrating the template was removed effectively. In addition, there were absorption bands in the both curves at around 1075 cm-1, which stood for the stretching vibration of Si-O-Si. UV−vis spectra of various growth processes of MSNR@Au shell further proved the successive formation (Fig. 2E). There is no plasmon peak was recorded for the initially formed MSNR (the blue curve), however for the MSNR@Au seeds (the red curve) there was a noteworthy peak around 515 nm. As the gold seeds grew, the peak displayed a red shift and became wider (the black curve). The changes of the peak positions demonstrated that with the gold seeds growing gradually, the distance 17

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between adjacent gold nanoparticles reduced.

Figure 2. Preparation and characterization of MSNR@Au shell. TEM images of (A) MSNR; (B) MSNR@Au seeds; (C) MSNR@Au shell; (D) Infrared spectroscopy of silica rod and silica rod removed CTAB; (E) The UV absorption spectra of various formulations to distinguish the formation of MSNR@Au shell. (F) (G) (H) (I) Energy-dispersive X-ray mapping images of MSNR@Au shell.

3.2. Synthesis and Characterization of MSNR@Au-TPPS4(Gd). XPS spectra (Fig. 3A, 3B) were utilized to verify that the HS-β-CDs could be linked to the surface of MSNR@Au shells through Au-S covalent bond. Both Au 4f and S 2p spectra exhibited broad signals, consistent with previously reported 47-48. As 18

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Fig. 3A showed, there were two peaks in Au 4f signal. The peak seated in 83.8 eV represented Au 4f7/2 and another seated in 87.5 eV represented Au 4f5/2, indicating the formation of the Au−S bond. Fig.3B showed a curve fitting spectrum through one pair of two doublets. In this picture, the binding energies of 2p3/2 were at 160.4 eV and 162.8 eV, which were attributed by monatomic sulfur anchored on the outside of MSNR@Au shells and the existence of polymeric sulfur species, respectively47,49. To construct versatile MSNR@Au-TPPS4(Gd) nanoparticles, MSNR@Au shells were modified with HS-β-CD by the Au−S bond to obtain MSNR@Au-β-CD. After that, the TPPS4(Gd) complex was non-covalently conjugated with MSNR@Au-β-CD through simply immersing the MSNR@Au-β-CD in TPPS4(Gd) aqueous solution to form stable inclusion complexes by host-guest interaction. As exhibited in the Fig.S1, the HS-β-CD/TPPS4(Gd) complex was triumphantly bound onto the surface of MSNR@Au shells with a wonderful rod-like morphology. The HS-β-CD/TPPS4(Gd) functionalization was also testified by DLS results, because the hydrodynamic diameter

was

changed

from

190

nm

(MSNR@Au

shells)

to

210

nm

(MSNR@Au-TPPS4(Gd)) (Fig.3E). Furthermore, the zeta potential of various formulations (MSNR, MSNR-NH2, MSNR@Au shell, and MSNR@Au-TPPS4(Gd)) during the reaction progress was −33.6, 30.7, -6.3, and −28.6 mV, respectively (Fig.3D). UV-vis spectra of MSNR@Au-TPPS4(Gd) nanoparticles (Fig.3C) displayed a small extra absorption peak at about 416 nm, meaning the formation of MSNR@Au-TPPS4(Gd). The final resultant HS-β-CD/TPPS4(Gd) coated MSNR@Au shells was obtained as MSNR@Au-TPPS4(Gd). The photosensitizer loading ratio was 19

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confirmed to be 17.77 wt% using UV-vis spectroscopy. Eventually, ICP−AES was utilized to measure the contents of gold and gadolinium in the MSNR@Au-TPPS4(Gd) and the molar ratio of gold to gadolinium is about 85. HS-β-CD has been used as nanocarriers for TPPS4 because of the host-guest interaction between TPPS4 and the HS-β-CD41. Interestingly, there still existed the fluorescence signal of TPPS4 after loading into MSNR@Au-CD (Fig.S2). Besides, the fluorescence spectra shown in Fig.S2 also certified that the fluorescence intensity of MSNR@Au-TPPS4(Gd) was lower and sharper than that of free TPPS4. In addition, as the raise of fed content of TPPS4, the drug-loading capacity increased and finally achieved the maximum value (57.2%) when the concentration of TPPS4 was 800 μg/mL (Fig.3F).

Figure 3. Preparation and characterization of MSNR@Au-TPPS4(Gd). (A) XPS spectrum of Au 4f area of MSNR@Au-β-CD. (B) XPS spectrum of S 2p area of MSNR@Au-β-CD. (C) The UV spectrum of various formulations to confirm the generation of MSNR@Au-TPPS4(Gd). (D) Zeta potential of MSNR, MSNR-NH2, MSNR@Au shell and MSNR@Au-TPPS4(Gd) nanoparticles, respectively. (E) Size distribution. (F) Loading capacity based on various TPPS4 concentrations. 20

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3.3. In Vitro MSOT/CT/MR Imaging. MSNR@Au-TPPS4(Gd) with different Au concentrations was monitored by MSOT images (Fig.4G). It was seen that MSOT images of the formulations were much brighter as the concentrations increased. The brighter of the samples, the stronger of the MSOT signal intensity. We also used quantitative analysis to demonstrate the relationship between the MSOT signal intensity and the Au concentrations. As the Fig.4G showed, the MSOT signal intensity was linear dependent with Au concentrations. For detecting CT imaging, the CT value exhibited a linearizing increase along with the gold concentration (Fig.4H). Phantom pictures of the MSNR@Au-TPPS4(Gd) at different Au concentrations in vitro were also obtained, which was consistent with the line. The ability of MSNR@Au-TPPS4(Gd) as contrast agents of MR imaging was also measured. In Fig.4I, the concentrations of Gd and the longitudinal (T1) relaxation time showed a linear correlation and the relaxivity r1 was 9.88 mM−1 s−1. These results were also showed in T1-weighted MR images. Furthermore, this result ascertained that the TPPS4(Gd) complex was triumphantly linked to the MSNR@Au-CD. As a result, MSNR@Au-TPPS4(Gd) could be used for in vivo multimodal imaging as a contrast agent with robustness from every in vitro imaging result. 3.4. In Vitro Photothermal Effect and ROS Detection. The Fig.4E exhibited that the temperature increased as the concentrations or irradiation duration increased, indicating an apparent photothermal effect of 21

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MSNR@Au-TPPS4 (Gd). In the meantime, digital thermometer and infrared thermal imaging camera were utilized to investigate the real-time temperature variations of different formulations. As shown in Fig.4A, D, the temperature elevations of MSNR@Au shell were similar with MSNR@Au-TPPS4 (Gd) solutions exposed to the same of continuous NIR laser, meaning that the photothermal effect of MSNR@Au shell was maintained after loading of TPPS4 (Gd). In comparison, there was little temperature rise in the PBS/H2O and free TPPS4 groups. In addition, the photothermal conversion efficiency was evaluated based on previous literatures50-51. Furthermore, the temperature curves of different MSNR@Au-TPPS4 (Gd) concentrations under 660nm or 808nm laser irradiation had been recorded (Fig.S3). We can see that the temperature increase of 660 nm (1W/cm2, 5 min) laser group was higher than that with 808 nm laser (1W/cm2, 5 min), but lower than that with 808 nm laser irradiation (1.5W/cm2, 5 min) (Fig.4E). From the literature52, the 808 nm laser was demonstrated that it had superior tissue penetration, so 808 nm laser was adopted as excitation light source to perform PTT effect. The temperature variations of the optimal formulation was detected for 300s followed by a cooling stage. The η of MSNR@Au-TPPS4 (Gd) was calculated to be 32.01% from the results (Fig.4B, C). Finally, there was no change in the temperature of MSNR@Au-TPPS4 (Gd) even after four irradiation cycles (Fig. S4). The results showed that MSNR@Au-TPPS4 (Gd) had excellent photothermal stability. In conclusion, MSNR@Au-TPPS4 (Gd) was a highly promising PTT nanoagent because of its NIR absorbance, outstanding photostability, and distinguished photothermal conversion efficiency. 22

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DPBF was employed as a trapper of ROS to investigate the ROS generation ability of MSNR@Au-TPPS4 (Gd) nanoparticles under 660 nm laser irradiation. According to the literature53, when there was 1O2 produced, the absorbency in ultra-voilet of DPBF at 410 nm would be decreased. As exhibited in Fig. 4F, the absorbency of the mixture, including DPBF and free TPPS4 (2 μg/mL), decreased to 32% of its raw data in 6 min. In the group of MSNR@Au-TPPS4 (Gd), there was an obvious decline of the absorbency of DPBF. However, there was no change of DPBF absorbance showed in either free DPBF or MSNR@Au shell-treated group, suggesting that MSNR@Au shell could not generate ROS. In addition, the decent degree of MSNR@Au-TPPS4 (Gd) was lower than that of free TPPS4 at the uniform conditions, which might because effective energy was absorbed by the MSNR@Au shell thus causing a loss of energy.

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Figure 4. PTT/PDT effect and MSOT/CT/MR imaging in vitro. (A)Temperature curves of various recipes under 808 nm irradiation (1.5 W/cm2, 5 min). (B) Photothermal effect of MSNR@Au-TPPS4 (Gd) under 808 nm irradiation (1.5 W/cm2). The irradiation continued for 300 s. (C) The linearity between cool-down time and negative natural logarithm of the temperature gotten from the cool-down section (after 300 s) in (B). (D) Thermographic images of PBS, MSNR@Au shell, and MSNR@Au-TPPS4 (Gd) under NIR laser (808nm, 1.5 W/cm2, 5 min). (E) The temperature curves of various MSNR@Au-TPPS4 (Gd) concentrations using 808 nm irradiation (1.5 W/cm2) for 5 min. (F) UV-vis absorbance changes of DPBF at 410 nm in various formulations under irradiation (660 nm, 1.0 W/cm2). (G) The linear relation of relative MSOT intensity and various Au concentrations. Inset: MSOT imaging pictures, including different concentrations of Au of MSNR@Au-TPPS4 (Gd) nanoparticles. (H) The CT numbers of the MSNR@Au-TPPS4 (Gd) solutions with different Au concentrations. Inset: the corresponding CT images of MSNR@Au-TPPS4 (Gd) nanoparticles. (I) Plot of 1/T1 versus various concentrations of gadolinium ion in MSNR@Au-TPPS4 (Gd) solutions. Inset: The corresponding T1-weighted MR pictures of MSNR@Au-TPPS4 (Gd).

3.5. Cellular studies. Before cellular studies, the stability of MSNR@Au-TPPS4 (Gd) nanoparticles was tested. The uniform concentrations of MSNR@Au-TPPS4 (Gd) nanoparticles were added into different solvents (Deionized water, PBS, DMEM and fetal bovine 24

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serum) and stored for 168 hours at 25°C. From Fig. S5, MSNR@Au-TPPS4 (Gd) nanoparticles exhibited excellent stability, therefore the formulation could be tested in cellular studies as well as in animal experiments. MTT method was utilized to verify the cytotoxicity of MSNR@Au-TPPS4 (Gd). Free TPPS4, MSNR@Au-β-CD and MSNR@Au-TPPS4 (Gd) with various concentrations were added into the 4T1 cancer cells respectively. From the Fig. 5A, cellular activity all maintained above 90% after 24 h cultivation, which indicated that MSNR@Au-TPPS4 (Gd) showed no obvious cytotoxicity

so

far

as

to

the

maximum

concentration

(163

μg/mL

of

MSNR@Au-β-CD and 35.4 μg/mL of TPPS4). Meanwhile, the hemolysis ratio was < 3% at the biggest denseness (200 μg/mL), and this suggested that the MSNR@Au-TPPS4 (Gd) nanoparticles had excellent biocompatibility, so it could be injected intravenously for antitumor treatment in vivo (Fig. S6). To testify the cellular PTT and PDT combined therapy, the irradiation of 660 and/or 808 nm were/was used to treat 4T1 cancer cells cultivated with different concentrations of free TPPS4, MSNR@Au-β-CD and MSNR@Au-TPPS4 (Gd). After that, the quantification of cellular activity was made. As shown in Fig. 5B, the ability of the MSNR@Au-TPPS4 (Gd) +808/660 nm killing cancer cells was dependent on the added dose. The cell viability of the 4T1 cells cultivated with the optimal formulation was decreased to 33% at the maximum concentration, lower than the 4T1 cells cultivated with free TPPS4 +660 nm or MSNR@Au-β-CD +808 nm at the same concentration. What’s more, co-staining research was used to exhibit the live (green, stained by calcein-AM)/dead (red, stained by PI) cells (Fig. 5C). Visibly, there is the 25

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strongest red color in the combination therapy group treated with MSNR@Au-TPPS4 (Gd) +808/660 nm compared with any other monotherapy group, suggesting the best antitumor efficacy of the optimal formulation. As revealed in Fig. 5D, significant red fluorescence signals could not be found in any group but the MSNR@Au-TPPS4 (Gd) +808 nm group. This result demonstrated the consumption of photosensitizer with the irradiation by 660nm or 808/660nm except for 808nm. However, MSNR@Au-TPPS4(Gd) could not produce ROS only under 808nm irradiation, as a result, it showed the red fluorescence as observed. There are much brighter green fluorescence signals in the MSNR@Au-TPPS4 (Gd) +808/660 nm group than that in the group of free TPPS4 +660 nm, which might because of the self-aggregation of free TPPS4 in aqueous environment. This phenomenon could not only result in the self-quenching of TPPS4 but also reduce the cellular uptake. MSNR@Au-TPPS4 (Gd) nanoparticles were proved that they were able to be taken in by 4T1 cells with no obvious cytotoxicity through all the results above. MSNR@Au-TPPS4 (Gd) nanoparticles could produce hyperthermia and ROS after being exposed to 808/660 nm laser irradiation to kill the cancer cells.

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Figure 5. (A) Cellular viability of 4T1 cancer cells cultivated with different formulations at kinds of concentrations. Data were showed as mean ± SD (n = 3). (B) Cellular viability of 4T1 cancer cells cultivated with kinds of concentrations of free TPPS4 + 660 nm, MSNR@Au shell-β-CD + 808 nm and MSNR@Au-TPPS4(Gd) +808/660 nm. Data were showed as mean ± SD (n = 3). (C) The pictures of stained 4T1 cancer cells cultivated with various groups. Scale bar: 100 μm. (D) The pictures of 4T1 cancer cells after being cultivated with various groups. Scale bar: 50 μm.

3.6. NIRF/MSOT/CT/MR Quadmodal Imaging in Vivo. In vivo NIR fluorescence microscopy was used to monitor the real-time in vivo distribution of the MSNR@Au-TPPS4 (Gd) nanocomposites (1.0 mg of TPPS4/kg) after being injected intravenously (Fig. 6A). Obviously, the fluorescence degree of the free TPPS4 group after administration for 12 h were strongest in the liver as well as the fluorescence signals suffered from attenuation inch by inch and nearly disappeared after 24 h injection. Conversely, after MSNR@Au-TPPS4 (Gd) injection 27

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for 4h, there were detected weak fluorescence at tumor sites. Then, the signals were gradually enhanced and finally up the maximum level at 12 h. And it should be noticed that there still was intense fluorescence signals in tumor sites at 24 h, indicating that MSNR@Au-TPPS4 (Gd) could reach at tumors with success and stay for an extended hours. In addition, we further measured the MSNR@Au-TPPS4 (Gd) distribution by ex vivo NIRF imaging. Tumors as well as major organs were taken out after 24 hours. Compared with more liver accumulation of free TPPS4, most TPPS4 in MSNR@Au-TPPS4 (Gd) was detected in the tumor rather than other organs, which kept consistent with the semiquantitative Fl intensity detection (Fig. 6B). The in vivo MSOT imaging, which integrated the merits of fine sensitivity and deep tissue penetration, was further assessed. Before and after the mice with tumor were injected with MSNR@Au-TPPS4 (Gd) solutions intravenously, MSOT imaging was performed. Evidently, the MSOT signals of MSNR@Au-TPPS4 (Gd) increased with time in the location of the tumor and were up the maximum value at 12 h after injection (Fig. 6C). Fig. 6D is the quantitative analysis, which exhibited that the MSOT signals of MSNR@Au-TPPS4 (Gd) at 12 h was 2.8 times compared with the pre-injected signals, implying the valid tumor accumulation of MSNR@Au-TPPS4 (Gd). As we know, gold had photoelectric effect to X-ray falloff because it’s a high atomic number element54. To evaluate the CT imaging capability of the MSNR@Au-TPPS4(Gd)

nanocomposites,

the

formulation

(25

mg/kg)

was

administrated into the mice with 4T1 cancer. There was an enhanced obvious contrast 28

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was observed after injection intratumorally (Fig. 6E). In addition, the CT values of Hounsfield units (HU) of the tumor sites were increased from 125 to 290 HU (Fig. 6F). Before and after MSNR@Au-TPPS4 (Gd) (1.27 μ mol Gd/kg) injection, T1-weight MR images were obtained using a 3T MR instrument because of the existence of TPPS4 (Gd) in the formulation. After intravenous injection for 12h, the tumor area showed a more lightful image than the pre-injection images (Fig. 6G), meaning a 689.3% rise of T1-weighted signal (Fig. 6H).

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Figure 6. NIRF/MSOT/CT/MR quadmodal imaging in vivo. (A) The NIRF pictures of mice with tumor at various point-in-time after being injected with free TPPS4 and MSNR@Au-TPPS4 (Gd) nanocomposites; the images at the bottom exhibits the fluorescence in the organs after 24 hours injection. (B) Relative intensity of fluorescent of TPPS4 in tumors and individual organs after being injected with free TPPS4 or MSNR@Au-TPPS4 (Gd) nanocomposites for 24 hours. (C) In vivo MSOT pictures of mice with 4T1 tumor after administration with MSNR@Au-TPPS4 (Gd) nanocomposites at various point-in-time intravenously. (D) The corresponding multispectral optoacoustic tomography intensity of MSNR@Au-TPPS4 (Gd) nanocomposites at different point-in-time in the tumor. (E) 3D (left)/ 2D (right) CT pictures of mice with 4T1 tumor before and after administration with MSNR@Au-TPPS4 (Gd). (F) The associated HU number of MSNR@Au-TPPS4 (Gd) nanoparticles before and after administration with MSNR@Au-TPPS4 (Gd). (G) T1-weighted MR pictures pre- and post-treatment of MSNR@Au-TPPS4 (Gd) nanocomposite.

(H)

The

corresponding

MR

intensity

MSNR@Au-TPPS4 (Gd) nanoparticles. (**) P < 0.01. 30

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and

post-treatment

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3.7. In vivo photothermal imaging In this study, the in vivo temperature variations were further monitored on the mice with 4T1 tumors. First, the mice with 4T1 tumors was injected intravenously with three different formulations respectively. Then, the laser (808 nm, 1.5 W/cm2) was utilized to radiate the tumor sites for 5 min after 12 h injection, and the temperature changes at specific time intervals were monitored by an IR thermal camera (Fig. 7). Just like we planned, after administration with MSNR@Au shell and MSNR@Au-TPPS4 (Gd), the mice temperature at the tumor surface of the quickly increased to 53.8 °C and 55.9 °C respectively, which meant that the photothermal effect of MSNR@Au shell maintained steadily after loading TPPS4 (Gd). At the same time, the temperature of the PBS groups showed a slight increase of approximately 6.7 °C in the irradiation process, demonstrating that MSNR@Au-TPPS4 (Gd) could be as an efficient PTT agents in vivo.

Figure 7. Thermographic pictures of mice with 4T1 tumors with exposure of the 808nm laser irradiation (1.5 W/cm2, 5 min) after injected with PBS, MSNR@Au shell and MSNR@Au-TPPS4 (Gd). The circles indicate the tumor.

3.8. In Vivo Antitumor Efficacy. 31

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Four treatments were designed to evaluate the PTT/PDT synergic anticancer efficacy of MSNR@Au-TPPS4(Gd) nanocomposites on the tumor-bearing mice. As shown in Fig. S7, the MSNR@Au-TPPS4(Gd) +808/660 nm groups inhibited the tumor growth notably compared with the group of the PBS. After treatments, the tumors were collected from four groups. As exhibited in Fig. 8A, the synergistic PTT/PDT exhibited the best treatment effect compared with any single therapy. In addition, we also the recorded the size of tumor volume, changes of mice weight and the rate of survivors for each group in the 21 treatment days (Fig. 8B, C and D). The PBS group demonstrated a higher body weight compared with other group, this maybe due to that the body weight could increase as the tumor increased. Besides, the MSNR@Au-TPPS4 (Gd) +808/660 nm group with an excellent antitumor effect exhibited no clear distinction in the changes of body weight compared with other groups, which indicated its great negligible toxicity and biocompatibility. Furthermore, we used H&E staining to evaluate the damage of tissues. As shown in Fig. 8E, MSNR@Au-TPPS4 (Gd) +808/660 nm could optionally damage the tumor tissues with no obvious damage to normal tissues compared with the PBS group. After being injected with MSNR@Au-TPPS4(Gd) for 24h, the major organs and tumors were obtained, followed by digestion in the aqua regia. After that, ICP-MS was employed to test the content of Au in the organs and tumors. From the results (Fig.S8), we could find that Au content was mainly distributed in liver (35.9% ID per g) and spleen (28.9% ID per g), which were the reticuloendothelial systems. Noticeable, Au nanoparticles were still accumulated in the tumor site (9.0% ID per g) 32

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even after 24h, demonstrating the good retention property of our formulation. The whole results demonstrated that MSNR@Au-TPPS4(Gd) under 808/660 nm laser irradiation exhibited a wonderful theranostic ability with no noticeable damage to normal organs.

Figure 8. (A) Pictures of the tumors gathered from various groups at the termination of treatment. (B) Percent survival of tumor-bearing mice in various treatment groups. (C) Relative tumor volume curves of mice with 4T1 tumors in various treatment groups. (D) Body weight changes of mice with 4T1 tumors in various therapeutic groups. (E) H&E stained pictures of individual organs and tumors gathered from the PBS group as well as the group with optimal formulation after 21 days treatment.

4. CONCLUSIONS 33

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In conclusion, MSNR@Au-TPPS4(Gd) theranostic nanocarrier with multi-modal imaging guided PTT/PDT synergistic anticancer therapy was successfully constructed. The nanocomposite combined rod-like gold shell with supramolecular PSs exhibited high photothermal conversion efficiency and superior ROS productivity, which not only provided NRIF/MSOT/CT/MR signals for quadmodal imaging-guided and real-time monitoring but also ensured the potential application for PTT and PDT combined treatment in the future. The combined hyperthermia/reactive oxygen species could induce outstanding cancer cell death under laser irradiation. The tumor targeting effect of MSNR@Au-TPPS4 (Gd) nanoparticles and excellent suppressive effect on tumor growth on the strength of the synergistic PTT/PDT anticancer therapy were also tested in vivo. In general, a novel multifunctional theranostic nanoparticle was assembled and it was meaningful in current-time imaging and imaging-guided combined anticancer effect.

Acknowledgments We acknowledge the financial supports from National Natural Science Foundation of China (Grant No. 81503016) and the National Basic Research Project (973 Program, 2014CB932200). ASSOCIATED CONTENT Supporting Information available: Characterizations, TEM images of MSNR@Au -TPPS4(Gd), fluorescence spectra, temperature curves of different MSNR@Au-TPPS4 (Gd) concentrations under 34

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different irradiations, photothermal conversion curve, dispersion stability, hemolysis, photos of 4T1 cell-bearing mice in the control group and the group treated with the optimal treatment plan, and biodistribution of Au in tumor sites and main tissues 24 h after intravenous administration of MSNR@Au-TPPS4(Gd) nanoparticles. References 1.

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DeSimone, J. M. The Effect of Particle Design on Cellular Internalization Pathways. Proc. Natl. Acad. Sci. U.S.A 2008, 105, 11613-11618. 2.

Mitragotri, S.; Lahann, J. Physical Approaches to Biomaterial Design. Nat Mater 2009, 8, 15-23.

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Interaction in Laminar Flows. Nanotechnology 2009, 20, 495101. 4.

Champion, J. A.; Katare, Y. K.; Mitragotri, S. Particle Shape: A New Design Parameter for

Micro- and Nanoscale Drug Delivery Carriers. J Control Release 2007, 121, 3-9. 5.

Decuzzi, P.; Godin, B.; Tanaka, T.; Lee, S. Y.; Chiappini, C.; Liu, X.; Ferrari, M. Size and Shape

Effects in the Biodistribution of Intravascularly Injected Particles. J Control Release 2010, 141, 320-327. 6.

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