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BSA-Bioinspired Gadolinium Hybrids Functionalized Hollow Gold Nanoshells for NIRF/PA/CT/MR Quad-modal Diagnostic Imaging Guided Photothermal/Photodynamic Cancer Therapy Qing You, Qi Sun, Meng Yu, Jinping Wang, Siyu Wang, Li Liu, Yu Cheng, Yidan Wang, Yilin Song, Fengping Tan, and Nan Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11926 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on October 31, 2017

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ACS Applied Materials & Interfaces 1

BSA-Bioinspired Gadolinium Hybrids Functionalized Hollow Gold Nanoshells for NIRF/PA/CT/MR Quad-modal Diagnostic Imaging Guided Photothermal/Photodynamic Cancer Therapy Qing You,† Qi Sun,† Meng Yu,‡ Jinping Wang,† Siyu Wang,† Li Liu,† Yu Cheng,† Yidan Wang,† Yilin Song,† Fengping Tan,† Nan Li*,† †

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

and Technology, Tianjin University, 300072 Tianjin, PR China ‡

PCFM Lab of Ministry of Education, School of Material Science and Engineering, Sun Yat-sen

University, 510275 Guangzhou, PR China

Corresponding author at: School of Pharmaceutical Science and Technology, Tianjin University, 300072, Tianjin, PR China. Tel: +86-022-27404986 E-mail address: [email protected]

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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ABSTRACT Multimodal imaging guided synergistic therapy promises more accurate diagnosis and higher therapeutic efficiency than single imaging modality or their simple “mechanical” combination. In this research, we constructed an innovative multifunctional drug delivery platform by gadolinium (Gd) based bovine serum albumin (BSA) hybrids coating hollow gold nanoshells (Au@BSA-Gd). The obtained nanoparticles exhibited excellent photothermal effect and computed tomography (CT)/photoacoustic (PA) activity. Besides, the BSA-bioinspired gadolinium complex endowed the nanoparticles excellent T1 contrast agent for magnetic resonance imaging (MRI). In addition, the NIR absorbing phototherapeutic agent (ICG) was loaded into the Au@BSA-Gd nanoparticles due to their unique hollow and porous structures, thus performing photodynamic/photothermal property and near-infrared fluorescence (NIRF)/PA imaging capability. As a result, a combined cancer therapy containing

photothermal

therapy

of

Au@BSA-Gd

and

synchronous

photodynamic/photothermal therapy of ICG was constructed. Furthermore, the well-designed nanocomposites with multiple integrated modalities enabled them to be an ideal nanotheranostic agent for NIRF/PA/CT/MR quadmodal imaging. Therefore, the ICG loaded albumin-bioinspired gadolinium hybrids functionalized hollow gold nanoshells (ICG-Au@BSA-Gd) hold great promise as a theranostic platform for simultaneously therapeutic monitoring and precise cancer therapy.

KEYWORDS: Hollow gold nanoshells; BSA-Gd hybrids; Indocyanine green; Quad-modal imagings; Photothermal therapy; Photodynamic therapy.


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1. INTRODUCTION In the past years, precision nanomedicines have attracted widely interest for etablishing theranostics which combine multi-modal imaging and therapeutic features.1-4 The preonunced informtion of the location of the tumor is important for specific cancer treatments.5, 6 To achieve this goal, a variety of nanoplatform are fabricated to accomplish accurate imaging modality such as near-infrared fluorescence (NIRF), magnetic resonance (MR), computed tomography (CT), positron emission tomography (PET) or photoacoustics (PA) for cancer monitoring7, 8 and further precise tumor therapy. However, single-modality imaging of nanostructures is hard to meet desired imaging requirements. For example, the NIR fluorescence (NIRF) probes with sensitivity suffer from poor resolution and shallow tissue penetration.9 In contrast, MRI offers an ideal spatial resolution but encounters poor sensitivity.10,

11

What’s more, CT imaging, which possesses high temporal

resolution and deep tissue penetration, still has difficulties to distinguish subtle changes of soft tissues.12,13 Multimodal imaging, however, can provide more accurate and wealthy information by compensating inherent limitations of each single imaging modality, thus precisely tracking the involved physiological and pathological process. Consequently, the rational design of a multifunctional all-in-one nanomedical platform system, which combines multimodal imaging diagnosis and therapy manners, is highly worthwhile to meet the growing demand for accurately destroying cancer and realizing precise therapy.14 Gold nanomaterials (GNP) have been extensively employed for cancer



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theranostic owing to their excellent stability and biocompatibility. Until now, various gold nanoparticles with tunable size and morphology, e.g., nanorods,15 nanoshells,16 and nanocages,17 have been widely applied for the thermal ablation of tumors due to their surface plasmon resonance (SPR) effect to convert the absorbed laser into heat, which can convert the absorbed laser into heat.18 This so-called photothermal therapy (PTT) of GNP upon the light irradiation has been primely constructed for tumor therapy. Apart from favorable therapeutic efficiency, the gold nanostructures also possess photoacoustic tomography feature and high X-ray attenuation coefficient, facilitating them as popular candidate for PA/CT imging.19, 20 Herein, we innovatively report a hollow porous gold nanoshells (HAuNs) based on the fragile mesoporous silicon as inner removable template.21 The unique properties of the porous structure and inner hollow space, integrating with the ease of the surface functionalization make the HAuNs a brilliant platform for the development of multimodal theranostic, by loading various functional materials such as organic dyes, chemodrugs or imaging agents.22 Recently, theranostic magnetic gadolinium-chelated gold nanoparticles have emerged for precise nanomedicine.23,

24

However, the existing methods for the

modification of gold nanoparticles with Gd3+ species suffer from shortcomings such as low Gd3+ loading capacity, undesired toxicity caused by easy Gd3+ leakage, and difficulties of further drug loading or surface functionalization.23, 25-26 With this in mind, we creatively fabricate the BSA-bioinspired Gd3+ hybrids functionalized hollow gold nanoshells (Au@BSA-Gd). As far as we know, the combination has not been


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reported before. We first utilized bovine serum albumin (BSA) to synthesize gadolinium-based hybrids by biomineralization method.27 Then the BSA-Gd complex could be coated outside the hollow gold nanoshells by amide bond linkage.28 Taking advantages of high longitudinal proton relaxivity and prolonged imaging time window of BSA-Gd, and

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this nanocarrier can be applied for simultaneous photothermal effect

MR/PA/CT

biomineralization

diagnostic outside

the

imaging.

In

hollow

gold

particular, nanoshells

the

albumin-based

exhibits

excellent

biocompatibility and good stability.27 Thereafter, considering the hollow inner cavities, antitumor efficiency and multimodal imaging for precise detection, indocyanine green (ICG), a tricarbocyanine dye with remarkable absorption in the NIR wavelength range,29 is incorporated into the Au@BSA-Gd nanoparticles for combined photothermal and photodynamic therapy (PDT). What’s more, ICG also exhibits strong near-infrared fluorescence (NIRF) and PA imaging.30 Noticeably, ICG is easy to aggregate and unstable in aqueous medium, which usually causes to the short half-life of plasma 31. Hence the problem can be well solved by loading into the Au@BSA-Gd nanocarriers. In this project, we demonstrate a multifunctional drug delivery system which synchronously integrates NIRF/PA/CT/MR quad-modal imaging and single light NIR-activated PTT/PDT effect (Figure 1). Our ICG loaded BSA-bioinspired gadolinium hybrids functionalized hollow gold nanoshells (ICG-Au@BSA-Gd) show several unique superiorities: (1) The inner void space and plentiful porous structures of the gold nanoshells ensure large amounts of various drug molecules, can be



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successfully encapsulated. (2) The ICG-Au@BSA-Gd nanoparticles show suitable stability and high biocompatibility under physiological conditions deriving from the BSA-Gd complex.27 (3) Compared with mono or binary modal imaging, the quad-modal imaging by the final formulations offers more accurate tumor information from a broad spectrum of intrinsic optical, electronic, and magnetic perspective with different imaging agents. (4) A combined therapy approach consisting of the PTT effect based on Au@BSA-Gd, as well as simultaneous PDT/PTT effect on account of ICG, can primely achieve the enhanced anticancer efficacy

2. MATERIALS AND METHODS 2.1. Materials. Ethanol, K2CO3, NaOH, Na2CO3, cetyltrimethylammonium bromide (CTAB) and tetraethylorthosilane (TEOS) were all purchased from Jiangtian Chemical

(Tianjin).

Chlorauric

N-[3-(trimethoxysily)propyl] (3-aminopropyl)triethoxysilane Indocyanine

green

acid

(HAuCl4),

ethylenediamine (APTES)

were

(ICG,

N-(3-(dimethylamino)-propyl)-3-ethylcarbodiimide

gotten

lipoic

acid,

(TSD) from

98%),

and

Sigma-Aldrich. Gd(NO3)3·6H2O,

hydrochloride

(EDC)

N-hydroxysuccinimide (NHS) were purchased from J&K Scientific Ltd.

and

BSA was

purchased from Solarbio. 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT),

1,3-Diphenylisobenzofuran

(DPBF),

dimethyl

sulfoxide

(DMSO),

4′,6-diamidino-2-phenylindoles (DAPI), propidium iodide (PI), calcein acetoxymethyl ester (Calcein AM) and Annexin V-FITC/PI apoptosis detection agents were obtained from Sigma-Aldrich. DCFH-DA probe was got from Tianjin Heowns Biochemical


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Technology Co., Ltd (China). High glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were all bought from Procell (China). 2.2. Preparation of Au@BSA-Gd Nanoparticles. 2.2.1. Preparation of Hollow Gold Nanoshells. Synthesis of fragile mesoporous silica (mSiO2): The hollow gold nanoshells were prepared by gold seed-mediated growth methods using the fragile mesoporous silica as a removable template. First, 45 mL deionized water containing 0.3 mL NaOH (2 M) was pour into a round bottom flask. When the temperature of the whole solution reached 70°C, a mixture of TSD and TEOS (600 µL) with a ratio of 1/4, 80 µL of APTES, and 3 mL of ethyl acetate were added in order. Then 80 µL of APTES was added after 10 min reaction two more times. The whole mixture solution was stirred for another 3 h under refluxing. Crude fragile mesoporous silica nanostructures were centrifuged (10000 rpm, 10 min) and then further washed with ethanol three times for collection. Then they were redispersed in ethanol (50 mL), heated to 80 °C, and refluxed overnight under stirring to remove CTAB. Finally fragile mesoporous silica were centrifuged (10000 rpm, 10 min), washed with ethanol more than three times, and then stored after lyophilization for further usage. Synthesis of mSiO2@Au seeds: First, 1 mL HAuCl4 (0.01 M) was added in 30 ml deionized water, then the pH value was regulated to 9.0 by adding 0.01 M NaOH to obtain a colorless transparent solution. 1.5 ml of the mSiO2 (about 7.5 mg) solution was added immediately for a further 30 min of intense stirring. In this procedure, HAuCl4 could attach on the surface of mSiO2 based on electrostatic adsorption. After



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that, 1.5 mL NaBH4 (0.01M) was added dropwise to reduce the HAuCl4. The resulting red solution was further stirred for another 6 h, separated by centrifugation, and eventually re-dispersed in 4 mL deionized water for further use. Synthesis of mSiO2@Au nanoshells and the final hollow gold nanoshells (HAuNs): potassium carbonate (K2CO3) (10 mg) was dissolved in 40 mL of deionized water then the obtained solution stirred for 10 min. Another portion of 1.5 mL HAuCl4 (0.01 M) was re-added for 30 min stirring until the yellow color of the solution became colorless. To this vigorously stirred solution, 500 µL mSiO2@Au seeds solution and 500 µL (78.8 mM) of L-ascorbic acid solution was added orderly. After reacting for 2 minutes, the solution emerged into blue green color, indicating the successful formation of Au nanoshells (Different amounts of mSiO2@Au seeds were added in the former gold seeds growth solution to explore the growth process of gold nanoshells). The mSiO2@Au nanoshells nanocomposites were centrifuged (10000 rpm, 10 min) and further purified by washing with deionized water. Subsequently, the mixture was placed into Na2CO3 solution (0.6 M) for etching 2 h under 90°C. Finally, the HAuNs were obtained by centrifugation and re-dispersed in 10 mL deionized water for further use. 2.2.2. Preparation of the COOH-HAuNs. The COOH-HAuNs was prepared by a typical procedure reported previously.24 The HAuNs were dispersed in 20 mL ethanol solution, then ten times weight of the lipoic acid were injected for 12 h reaction. Afterward, the COOH-HAuNs were ` by centrifugation (10000 rpm, 10 min) and purified by washing with ethanol for several times.


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2.2.3. Preparation of BSA-Gd Complex. BSA-Gd complex was prepared by the previously reported protocol.27 Briefly, BSA (0.25 g) was injected into 9 mL of deionized water at 37 °C, then 1 mL Gd(NO3)3 aqueous solution (50 mM) was slowly added. After 5 min, 1 mL NaOH (2 M) was quickly added into the former mixture and reacted at 37 °C for 12 h. Thereafter, the solution was poured into a dialysis bag (cutoff Mw = 3500) and dialyzed against deionized water for 24 h to purify the product. The BSA-Gd complex were collected and stored in a refrigerator. 2.2.4. Preparation of Au@BSA-Gd Nanoparticles. For preparing BSA-Gd modified HAuNs, 3 mg of COOH-HAuNs were ultrasonically dispersed in 30 mL of deionized water, and then 5 mg of EDC and 3.6 mg of NHS were introduced into the solution to activate the carboxyl group. After being stirred for 2 h, 0.5 mL of BSA-Gd complex was introduced into the above system and reacted for 1 day. The obtained Au@BSA-Gd nanoparticles were harvested and purified by centrifugation. 2.3. ICG Loading and Subsequent Release Behaviors. In ICG loading studies, 1mL various concentrations (50, 100, 200, 300, 400, 500µg/mL, respectively) of ICG solution were mixed with Au@BSA-Gd (1.0 mg) PBS solutions (pH 7.4) and further reacted about 8 h. The obtained formulations were centrifuged, and purified by washing with PBS several times to isolate free ICG. For investigating the ICG loading ratio, all of the supernatant in the different washing processes were gathered and then measured the absorbance at 780 nm. The ICG loading ratio was expressed as: Loading ratio = (MICG－MuICG)/MAu@BSA-Gd ×100% where MICG, MuICG and MAu@BSA-Gd were the total amounts of ICG, unloaded ICG and



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Au@BSA-Gd, respectively. The release behaviors of ICG were investigated by the dialysis method at 37 °C in different PBS (pH 7.4, 6.5, and 5.0) solution. 4 mL of ICG-Au@BSA-Gd was placed in a dialysis bag and then immersed into 40 ml PBS receptor fluid. For each predesigned time interval, samples (2 ml) from the PBS were taken and detected by using the UV/vis spectrum (780 nm) to quantify the amount of released ICG. At the same time, 2 mL of new PBS solution was further introduced to maintain a consistence. All the release studies were operated in a dark environment. 2.4. Characterizations of ICG-Au@BSA-Gd Nanoparticles. Transmission electron microscopy (TEM) images were gotten by a JEM-100CX electron microscope (JEOL Ltd., Japan). The elemental mapping images were captured by using a high resolution TEM (HRTEM). (JEM-2100f, Japan). The surface morphology was detected by scanning electron microscope (SEM, Hitachi, SU8020, Japan). The size distribution and zeta potential of various nanoparticles were investigated by dynamic light scattering (DLS) with Zetasizer Nano ZS (Malvern, UK). UV-vis absorption spectrums were determined by a Cary 60 UV-vis spectrophotometer (Agilent, USA). The FT-IR spectra were measured on a TESOR-27 spectrophotometer (Bruker, Germany). The circular dichroism (CD) spectra were recorded by a spectropolarimeter system (Jasco J-810, Ltd., Japan). The fluorescence intensity was performed on an LS-55 fluorescence spectrophotometer (Perkin-Elmer). The concentration of Au and Gd was investigated on inductively coupled plasma atomic emission spectroscopy (ICP-AES) (7700x, Agilent). Laser irradiation was


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conducted using a semiconductor power-tunable laser (LASERGLOW Technologies, Shanghai, China). The solution temperature was gauged with a TASI-8620 digital thermometer (TASI, China). Thermal images were recorded on a TI400 infrared camera (Fluke TiR, USA). 2.5. In Vitro PA/CT/MR Imaging of ICG-Au@BSA-Gd Nanoparticles. In vitro photoacoustic effect of the ICG-Au@BSA-Gd nanoparticles was tested by a photoacoustic imaging system. The aqueous solutions of the formulation with different ICG-Au@BSA-Gd concentrations (Here different concentrations of ICG-Au@BSA-Gd

was

presented

with

the

corresponding

different

ICG

concentrations) were added into the tube to measure the PA signals on a Vevo LAZR PAI system at the wavelength of 808 nm, respectively. The ICG-Au@BSA-Gd nanoparticles dispersions (in PBS) with different Au concentrations (0, 18.75, 37.5, 75, 150, 300 µg/mL, respectively) were prepared in tubes (1.5 mL) and carried out by small mice X-ray CT (Gamma Medica-Ideas). In vitro MRI studies were detected by a 7T MRI system (Varian, US). T1-weighted

images

of

ICG-Au@BSA-Gd

nanoparticles

at

various

Gd3+concentrations (0.025, 0.05, 0.1, 0.2, 0.4 mM, respectively) in deionized water were obtained. Here are the relative parameters: FOV read = 34 mm, FOV phase = 40 mm, repetition time (TR)/effective time (TE) = 3500 ms/72 ms, slices = 20, slice width = 20 mm, slice gap = 0 mm. 2.6. Measurement of Photothermal Performance. Different concentrations of Au@BSA-Gd aqueous dispersions (0.5 mL) and various samples (0.5 mL) (PBS,



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BSA-Gd, Au@BSA-Gd, and ICG-Au@BSA-Gd) were placed in the centrifuge tubes and exposed to the NIR laser (808 nm, 1.5 W/cm2, 5 min). The solution temperature changes were measured with a digital thermometer at pre-designed time intervals. The thermal images of different formulations were also captured by using an infrared thermal camera. For evaluating the photothermal conversion efficiency (η), the temperature changes of ICG-Au@BSA-Gd (with Au@BSA-Gd concentration of 100 µg/mL and ICG concentration of 10 µg/mL, respectively) dispersion were measured under a successive laser irradiation (808 nm, 1.5 W/cm2) until the temperature of the formulation didn’t change. The η value was acquired by the following formula: η=

hS(Tmax-TSurr) - QS ×100% I (1-10-A808)

where h represents the heat transfer coefficient, S is the surface area of the vessel. Tmax expresses the supreme temperature, TSurr is the surrounding temperature, QS represents the heat associated with the laser absorbance, I expresses the incident light power and A808 represents the absorbance value of the optimal dispersion at 808 nm laser. 2.7. Stability Studies of the ICG-Au@BSA-Gd. The storage and dispersion stability of ICG-Au@BSA-Gd was first investigated. Samples containing the same amount ICG-Au@BSA-Gd were added to deionized water, PBS, and Dulbecco’s Modified Eagle’s Medium (DMEM) medium, containing 10% fetal bovine serum (FBS), respectively. The different formulations were kept at room temperature (25°C) and observed for 168 h. Picture and absorption spectra of the different formulations


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were recored at 1 h, 24 h, and 168 h. For testing the photostability, ICG-Au@BSA-Gd was measured by spectrum analysis of the solution under NIR laser irradiation. Samples containing free ICG (5 µg/mL), and ICG-Au@BSA-Gd (ICG: 5 µg/mL; Au@BSA-Gd: 50 µg/mL) were irradiated under NIR laser (808nm, 1.5 W/cm2) for 0, 2, 4, 6, 8 and 10 min, respectively. The spectra were recorded immediately after the irradiation. The percentage of residual ICG was calculated by comparing with the original supreme intensity of the samples. Photothermal cycling tests were also conducted. 0.5 mL of ICG-Au@BSA-Gd solution was irradiated with four on-off laser irradiation (808nm, 1.5 W/ cm2) and then temperature variations were recorded. 2.8. Determination of In Vitro ROS. The generation of 1O2 was assessed by using 1, 3-diphenylisobenzofuran (DPBF) as 1O2 sensor. 2 mL ICG (5 µg/mL), Au@BSA-Gd (50 µg/mL) and ICG-Au@BSA-Gd (50 µg/mL for Au@BSA-Gd and 5 µg/mL for ICG, respectively) were mixed with 20 µL of DPBF (5 mmol/L, DMSO), respectively. The DPBF absorbance (at 410 nm) in the above mixtures was measured before and after laser irradiation (808 nm, 1.5 W/cm2) at indicated time points. 2.9. Cellular Culture and Cellular Uptake Studies. Murine breast cancer cell line (4T1 cells) provided by Procell Life Science & Technology Co., Ltd (Wuhan, China) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, GIBCO, Invitrogen) complete medium, containing 10% fetal bovine serum (FBS), 100 U/mL penicillin or 100 µg/mL streptomycin. The culture atmosphere is at 37 °C with a humidified condition with 5% CO2. To visualize the cellular uptake of ICG-Au@BSA-Gd nanoparticles, 4T1 cells (5



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× 105 cells/well) pre-seeded in CLSM culture dishes were incubated with free ICG and ICG-Au@BSA-Gd (with the same ICG concentration of 10 µg/mL) for 4 h, respectively. Then cells were washed by PBS solution, stained with DAPI and measured on a confocal laser scanning microscope (CLSM, Leica Microsystems, Heidelberg, Germany). 2.10. Detection of Intracellular ROS. Dichlorofluorescein diacetate (DCFH-DA) was chosen to assess intracellular reactive oxygen species produced by ICG-Au@BSA-Gd and ICG. 4T1 cells were seeded into the CLSM culture dishes at a density of 1 × 106 cells per well incubated with various formulations (PBS, free ICG, Au@BSA-Gd and ICG-Au@BSA-Gd, respectively) for 12 h. After washed with PBS, the cells were cultured with DCFH-DA (10 µM at 37 °C for 50 min, washed again and irradiated by light irradiation (808 nm, 1.5 W/cm2, 3 min). DCFH in cytoplasm can be oxidized to a highly fluorescent derivative in the presence of reactive oxygen species32 and then visualized by using a CLSM (DAPI, 360/460 nm; DCF, 488/525 nm; ICG, 633/700 nm). 2.11. In Vitro Cytotoxicity Evaluations. MTT assay was utilized to identify the cellular toxicity of designed formulations. 4T1 were pre-seeded into a 96-well plate (5×104 per well) and then incubated with various concentrations for 24 h in dark. For phototoxicity, cells were irradiated by an NIR laser (808 nm, 1.5 W/cm2, 5 min) after 6 h incubation and further incubated for 18 h. Thereafter, we injected 20 µL of MTT solution to each well and further incubated for 4 h at 37 °C. Then the culture media were separated, and 500 µL of DMSO was added to dissolve the formazan crystals.


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The absorbance at 490 nm of each well was tested using a microplate reader. The tumor cell-killing ability was further evaluated by Live/Dead cell costaining studies. 4T1 cells (1 × 106) were pre-seeded into CLSM dishes and then incubated with various formulations (10 µg/mL of ICG and 100µg/mL of Au@BSA-Gd) for 12 h with NIR laser (808 nm, 1.5 W/cm2, 5 min). After washing the cells by PBS for several times, the calcein-AM (4 µM) and propidium iodide (PI) (4 µM) were orderly introduced for further incubation about 20 min. Eventually cells were washed several times, and imaged immediately by CLSM (calcein-AM, 488/515 nm; PI, 535/617 nm). For flow cytometry, 4T1 cells (1 × 106 cells per well) were cultured in a 6-well plate for 24 h. The cells then were incubated with various formulations (10 µg/mL of ICG and 100 µg/mLof Au@BSA-Gd) for 12 h with NIR laser irradiation (808 nm, 1.5 W/cm2, 5 min). Afterward, the cells were detached, collected, and repeatedly washed with PBS by centrifugation. After being filtered, the cells suspended in PBS were analyzed by flow cytometer (BectonDickinson, U.S.). 2.12. Animals and Tumor Models. All the nude mice and Kunming mice were bought from Huafukang Biological Technology Co. Ltd (Beijing, China). All animal experimental procedures were operated in adherence with a standard protocol approved by the Institutional Animal Care and Use Committee of Tianjin University. Tumor models were established by injecting 4T1 cells (3 × 106), subcutaneously into the oxter parts. Animal studies were performed when tumor volume reached 100 mm3. 2.13. In Vivo Quad-Modal Imaging. For in vivo fluorescence imaging, nude



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mice bearing 4T1 cells were injected with 200 µL of free ICG and ICG-Au@BSA-Gd (ICG, 1.0 mg/kg) respectively through a lateral tail vein. After injection, time-dependent fluorescence images were measured using the IVIS Lumina imaging system (Caliper, USA) at an excitation and emission wavelength of 735 nm and 780-950 nm, respectively. After in vivo imaging, the mice were executed and the major organs integrated with tumors were taken out for ex vivo imaging and quantitative analysis. In vitro fluorescence signals of the released ICG at different time points were also investigated on the IVIS Lumina imaging system (Caliper, USA). Briefly, 4 mL of ICG-Au@BSA-Gd was placed in a dialysis bag and then immersed into 40 ml PBS receptor fluid. For each predesigned time interval (1h, 6h, 12h and 24h), samples (2 ml) from the PBS were taken and visualized for ICG signal. At the same time, fresh PBS solution of the same volume was introduced to keep a consistence. All the release studies were operated in a dark environment. For in vivo PA imaging, tumor-bearing nude mice were intravenously administrated with 200 µL Au@BSA-Gd and ICG-Au@BSA-Gd nanoparticles (ICG, 1.0 mg/kg) with the same amount of Au@BSA-Gd. Then the PA images were captured at pre-designed time points (0 h, 1 h, 6 h, 12 h and 24 h). PA signal intensity was then determined using Image J. For in vivo CT imaging, 200 µL of ICG-Au@BSA-Gd nanocomposites (25 mg/ kg) were first administrated into tumor-bearing nude mice. Then the mice were anesthetized and imaged on the X-ray CT equipment (Gamma Medica-Ideas) for imaging before and after injection. Relative parameters: voltage, 80 kV; pixel size, 50


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µm; current, 270 µA; field size, 1024 pixels × 1024 pixels. For in vivo MR imaging tests, 4T1 tumor-burdened nude mice were injected with ICG-Au@BSA-Gd nanoparticls (0.05 mmol Gd/kg mice) and T1-weighted signals were recorded before and 12 h after injection using a 7T MR imaging system (Varian, US). The detailed parameters were same with the in vitro MR imaging. 2.14. In Vivo Blood Circulation and Biodistribution. To measure the blood circulation, 4T1 tumor-burdened nude mice were intravenously injected with ICG-Au@BSA-Gd nanoparticls (10 mg/ mL, 100 µL). Then we gathered the blood samples (200 µL) at pre-designed time points, further weighted and solubilized them by nitrohydrochloric acid. Next the Au contents of the processed solutions were investigated at pre-designed time points by ICP tests to obtain the bloodstream pharmacokinetics profile of ICG-Au@BSA-Gd nanocomposites. For biodistribution measurement, mice were sacrificed after 24 h intravenous injection of ICG-Au@BSA-Gd nanocomposites (10 mg/mL, 100 µL). Then we gathered the Organs and tissues, further weighted and dissolved them by nitrohydrochloric acid. Afterwards, the contents of Au and Gd were tested by ICP. The contents of ICG-Au@BSA-Gd were expressed using the percentage of given dose per gram of tissue (%ID /g). 2.15. Intratumoral PTT/PDT Effects. To evaluate the NIR-induced in vivo photothermal effect, nude mice after intravenously administered with different formulations (PBS, ICG, Au@BSA-Gd and ICG-Au@BSA-Gd, respectively) 12 h, were given a laser irradiation (808nm, 1.5 W/cm2, 5 min) in the tumor sites. The



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18

infrared thermographic images of the tumor sites were recorded with an infrared thermal camera. NIR-triggered intratumoral ROS production was determined using DCFH-DA as a probe. Briefly, tumor-bearing mice were treated with PBS, ICG+NIR, ICG-Au@BSA-Gd, and ICG-Au@BSA-Gd+NIR, respectively. After 12h, the mice were executed, then tumors tissues were isolated, embedded, and cut into 8-µm slices. After incubation with DCFH-DA for 20 min, cell nuclei were stained by DAPI. Finally, samples were observed immediately with confocal microscopy. 2.16. In Vivo Antitumor Efficiency. To test the antitumor effect, 4T1 tumor-bearing female Kunming mice were randomly divided into five groups after tumor volume was about 100 mm3: (1) PBS, (2) PBS+NIR; (3) Au@BSA-Gd+NIR; (4) Free ICG + NIR (5) ICG-Au@BSA-Gd+NIR. Tumor volume and body weight of the mice before and after treatment were evaluated using a caliper and an electronic balance. The tumor volume can be calculated based on the normal equation (volume = width2 × length/2) commonly used in previous reports.33 What’s more, the survival curves were also recorded after the therapies. On the last day, tumors and organs were isolated, and further performed with hematoxylin and eosin (H&E) staining studies. 2.17. Statistical Analysis. Statistical analysis was used as mean ± standard deviation (SD) and conducted by ANOVA. P values were obtained using the 2-sample t-test. The probability level of 95% (p < 0.05) represents significant difference.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of HAuNs. The hollow gold nanoshells


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(HAuNs) were innovatively prepared based on the fragile mesoporous silicon (mSiO2) as inner removable template (Fig.2a). The vulnerable mSiO2 was synthesized by insertion of TSD, which made the hybrid silica layer loose and sensitive to alkaline solution.34 According to the so-called one-pot in situ Au3+ reduction method,16 Au3+ were attached to the surface of mSiO2 by electrostatic adsorption and then locally reduced to form gold seeds anchoring mSiO2 nanoparticles (mSiO2@Au seeds). Subsequently, the gold seeds grew bigger and became continuously porous gold nanoshells (mSiO2@Au shells), by precisely controlling the following seed-mediated growth. The final unique HAuNs were obtained by etching the inner silica in sodium carbonate solutions. Themorphologies and structures of the hollow gold nanoshells coated mSiO2 during the different growth processes and the obtained hollow gold nanoshells were measured by transmission electron microscopy (TEM). Figure 2bI showed that the fragile mSiO2 nanoparticles had a well-defined spherical structure the diameter about 120 nm and high dispersity; Figure 2bII exhibited the gold seeds were triumphantly grown on the outer surface of mSiO2 (mSiO2@Au seeds), and still maintained well-dispersed. Figure 2b III–IV demonstrated the seed-mediated grow processes. Gold seeds progressively grow larger, and eventually the nanoshells capped mSiO2 (mSiO2@Au shells) with spherical morphology and excellent dispersity were obtained (Figure 2bV). After etching the inner silica materials for 2 h, the unique hollow porous gold structures were obtained (Figure 2bVI). UV-vis spectra and the color changes of HAuNs of various growth processes further proved the continuous



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20

formation. For initially formed mSiO2 (Figure 2c), no plasmon peak was recorded (curve 1). While there was an obvious peak around 510 nm when coated with the gold seeds (curve 2). With gradual growth of gold seeds, the peak became wider and displayed a red shift (curve3 to 5). The evolutions of the peak positions vertified distance of the adjacent gold particles decreased owing to progressive growth of the gold nanoparticles, which were consistent well with the TEM data. Curve 6 exhibited a strong and wide absorption in the NIR region, indicating the extensive contact of Au particles and the successful formation of mSiO2@Au nanoshells after the gold nanoparticle’s progressive growth. What’s more, after etching the inner mSiO2, the HAuNs still showed a strong absorption (as curve7), which was a key to photothermal therapy. In addition, the growth stages were further recorded by measuring the color changes of the different formulations, which was consistent with the UV-vis spectra (Figure 2d). The initial mSiO2 displayed white color. Then the color of the solution became purple after the increase of HAuCl4 solution, on account of surface plasmon resonance of the gold nanoparticles. Upon further seed-mediated growth, the color of the formulation became into deep purple, then blue, and eventually blue-green. What’s more, the final HAuNs also showed a blue-green color with no difference with mSiO2@Au nanoshells. Therefore, the HAuNs growing process was controllable, thus providing well-tuned optical properties. The structures of HAuNs were further confirmed by SEM images (Figure 2f) with a significant rough surface compared with the mSiO2. Moreover, the element mapping analysis (Figure 2e) revealed that Au (yellow) distributed in the outer layers.


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As expected, the inner silicon was obviously reduced and almost distributed annularly along with the outer gold nanoshells, suggesting the successful removal of the brittle silica layer without damage of gold nanoshells. The ringlike distribution of silicon was probably attributable to the residual silicon elements on the gold nanoshells during their dissolution after etching. Thus, the obtained extra inner space was able to potentially apply for other therapeutic agents. 3.2. Synthesis and Characterization of Au@BSA-Gd Nanoparticles. To construct versatile Au@BSA-Gd nanoshells, the HAuNs were first modified with lipoic acid by Au-S bond to achieve COOH-HAuNs. Subsequently, BSA-Gd complex was

covalently

conjugated

onto

the

surface

of

COOH-HAuNs

through

carbodiimide-catalyzed amidation reaction (Figure 3a).As displayed in the TEM images (Figure 3b), BSA-Gd complex was successfully conjugated outside the surface of HAuNS with an excellent spherical morphology. The DLS results also confirmed the BSA-Gd functionalization as the hydrodynamic diameter was increased from125.3 nm (HAuNS) to 151.1 nm (Au@BSA-Gd) (Figure 3c). Besides, the zeta potential of different formulations (HAuNS, COOH-HAuNs and Au@BSA-Gd) during the synthetic process was -24.3 mV, -36.3 mV and -16.7 mV, respectively (Figure S1).

Fourier transform infrared spectroscopy (FTIR) and circular dichroism

(CD) characterizations were further adopted to test the linkage of BSA-Gd by identifying the BSA typical spectra. As shown in Figure 3d, the Au@BSA-Gd nanoparticles, consistent well with pure BSA and BSA-Gd hybrids, displayed characteristic absorption around 1650 and 1541 cm−1 which belonged to the amide I



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22

and amide II bands of BSA,35 respectively, suggesting the successful attachment of BSA-Gd onto COOH-HAuNs. Meanwhile, both of BSA-Gd and Au@BSA-Gd nanoparticles had significant peak at around 210 nm with slight blue shift (Figure 3e), compared to the CD spectra of pure BSA. This also verified the elaborate construction of Au@BSA-Gd and the slight blue shift rooted in increased random coil structures.36 Last, the molar ratio of Au to Gd in Au@BSA-Gd was measured to be ~8.15 with ICP-AES. The high loading content of Gd was expected to confer Au@BSA-Gd ability to enhance the contrast in MR images. 3.3. ICG Loading and Subsequent Release. The serum albumin has been used as nanocarriers for ICG due to the hydrophobic interaction between ICG and the hydrophobic domain of BSA.37 Here on basis of the inner hollow structure of Au@BSA and the outer BSA structures, the final formulations were able to potentialy apply for loading ICG molecule. Figure 3f revealed the color of the Au@BSA-Gd dispersed solution changed from blue to green after loading ICG. Besides, the UV-visble spectra shown in Figure 3g demonstrated the absorption peak of ICG-Au@BSA-Gd was higher than Au@BSA-Gd and broader than free ICG. In addition, the maximum absorption peak of the optimal formulations exhibited an obvious red shift compared with free ICG and the physical mixture of ICG and Au@BSA-Gd, stating the successful ICG loading. Interestingly, it was found that the fluorescence signal of ICG was obviously quenched after loading into Au@BSA-Gd (Figure S2). What’s more, the drug loading capacity increased with the raise of fed content of ICG and eventually reached the highest value (36.5%) when ICG


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concentration was about 400 µg/mL (Figure 3h). Moreover, there were no obvious differences of the ICG release profile among pH 5.0, 6.5, and 7.4, illustrating that the pH value barely affected the release behavior (Figure 3i). 3.4. Stability Studies of ICG-Au@BSA-Gd Nanoparticles. Dispersion stability of the ICG-Au@BSA-Gd complex dispersed in different mediums (deionized water, PBS, and cell culture media containing 10% FBS) has been tested. As exhibited in Figure S3, the ICG-Au@BSA-Gd nanoparticles exhibited excellent dispersity and no aggregation of all solutions during 7 days’ storage. Considering the unstable optical properties of ICG poor aqueous stability and concentration dependent aggregation properties, we investigated the stability of free ICG and the optimal formulations. The results showed the absorbance of free ICG decreased significantly after 10 min irradiation, while no obvious changes could be observed of the ICG-Au@BSA-Gd solutions (Figure S4), proving the outstanding photostability of the optimal formulations. 3.5. In Vitro PA/CT/MR Multimodal Imaging Test. ICG-Au@BSA-Gd with various ICG concentrations was detected by photoacoustic (PA) images and presented in Figure 4a. A quantitative analysis demonstrated a linear correlation between the PA signal intensity and the ICG concentrations ranging from 0 to 60µg/mL, showing final formulation-concentration dependent profile. To assess the ability for CT imaging, the CT contrast was evaluated by acquiring the phantom images of the optimal formulations at different concentrations in vitro (Figure 4b). It was found that the CT images of the dispersions became progressively



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24

brighter along with the increasing concentrations, indicating the incremental CT signal intensity. The calculated CT value revealed a linear increase with the Au concentration, showing a relatively high X-ray absorption coefficient. For monitoring the capacity of ICG-Au@BSA-Gd as effective T1-weighted MR imaging contrast agents, the longitudinal (T1) relaxation times were measured. As shown in Figure 4c, T1-weighted MR images and their corresponding signal intensity exhibited a linearly Gd-concentration-dependent increased MRI characteristics with the relaxivity r1 of 10.05 mM-1s-1, stating a satisfied value for Gadolinium based MR contrast agents. This result further confirmed that the BSA-Gd complex was successfully anchored to the HAuNs. In addition, the final formulation could act as highly efficient T1-weighted MR imaging contrast agents for biomedical imaging. All of the in vitro imaging results indicated the robustness of ICG-Au@BSA-Gd as a contrast agent for in vivo multimodal imaging. 3.6. In Vitro Photothermal Effect and Reactive Oxygen Species Detection. In order to explore the photothermal effect of this therapeutic paltform, the temperature increase was examined upon a laser irradiation (808 nm, 1.5 W/cm2, 5min). As seen from Figure 4d, the photothermal effect of Au@BSA-Gd was pronounced, which showed a concentration- and irradiation duration-dependent temperature increase manner. Meanwhile, the real-time temperature changes of different formulations were also investigated with a digital thermometer and infrared thermal imaging camera. As indicated in Figure 4e and 4j, continuous NIR laser exposure resulted in similar temperature elevations of free ICG and Au@BSA-Gd solutions. In comparison, only a


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slight temperature change was detected in PBS and BSA-Gd group. Moreover, the ICG-Au@BSA-Gd suspensions proved the higher NIR thermal imaging properties under the same irradiation conditions, revealing the significantly enhanced PTT effect by the combination of ICG and Au@BSA-Gd. The photothermal conversion efficiency was further evaluated according to a previous report.38,

39

The sample

temperature change curve was recorded as a function of continuous irradiation time until the temperature of the solution didn’t change. The obtained results (Figure 4g, 4h) indicated the photothermal conversion efficiency of ICG-Au@BSA-Gd was finally calculated as 21.77%. Eventually, it was also found that the temperature promotion (Figure 4f) of the ICG-Au@BSA-Gd exhibited unchanged after four repeated irradiation cycles, indicating satisfactory photothermal conversion stability. The strong NIR absorbance, high photothermal conversion efficiency and excellent photostability made the ICG-Au@BSA-Gd highly promising as a PTT nanoagent. To

confirm

the

singlet

oxygen

generation

(SOG)

capability

of

ICG-Au@BSA-Gd, 1, 3-diphenyl isobenzofuran (DPBF) was used as a singlet oxygen trapping agents, whose absorbance could be quenched by forming DPBF-endoperoxide compounds.40 As a result, the DPBF absorbance decreased dramatically to 24.3% of its original value under 808 nm laser, which indicated that ICG-Au@BSA-Gd could efficiently generate singlet oxygen upon photoirradiation (Figure 4i). What’s more, the ROS production of the optimal formulations was much slower and lower than free ICG (decreased to 2.3% of its original value) under the same conditions owing to a loss of effective energy absorbed by the Au@BSA-Gd to



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26

perform PTT effect at the same time. 3.7. Cellular Uptake Study and Intracellular ROS Detection. Successful cellular uptake and internalization of nanoparticles by tumor cells are important in anti-cancer therapy. To test the cellular uptake of the ICG-Au@BSA-Gd, 4T1 cell were incubated with PBS, ICG and ICG-Au@BSA-Gd, respectively. Confocal microscopy expressed significant red fluorescence in the cytoplasm after incubation with ICG-Au@BSA-Gd, suggesting the efficient cellular uptake and intracellular distribution (Figure S5). What’s more, the fluorescence intensity of the final formulation exhibited clearly stronger compared with free ICG under the same conditions, which might be attributed to their enhanced uptake and reduced degradation after loaded into the Au@BSA-Gd nanocarriers. Then, we investigated intracellular ROS generation, using nonfluorescent dichlorofluorescein diacetate (DCFH-DA) as fluorogenic probe.41 When there exist ROS, DCFH-DA could be oxidized and altered into dichlorofluorescein (DCF), which could show obvious green fluorescence. According to results shown in Figure 5c, no significant green fluorescence was detected in PBS and ICG-Au@BSA-Gd group. While in the ICG-Au@BSA-Gd plus NIR laser irradiation group, 4T1 cells exhibited stronger DCF fluorescence intensity, suggesting the excellent ROS generation capability. However, relatively weak fluorescence signals could be detected in the free ICG + 808 nm laser-treated cells, because of the poor uptake ability of free ICG. This result proved that ICG-Au@BSA-Gd nanomaterials could induce intracellular ROS generation with the NIR laser irradiation for PDT cancer therapy.


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3.8. In Vitro Single Light-Induced Cytotoxicity. To quantitatively elucidate the cytotoxicity and phototoxicity of the obtained formulations to 4T1cancer cells, the MTT assays were carried out. After tumor cells were incubated with different formulations (ICG, Au@BSA-Gd and ICG-Au@BSA-Gd) of various concentration for 24 h, no obvious lethal effect (cell viability was above 90%) was observed even at highest concentration (Figure 5a). The results indicated the good biocompatibility of the as-prepared ICG-Au@BSA-Gd nanocomposites, which was a prerequisite for biomedical applications of nanoagents. However, all of the treated groups displayed dose-dependent antitumor efficiency upon a laser exposure (808nm, 1.5 W/cm2, 5 min) (Figure 5b). What’s more, similar NIR illumination of ICG-Au@BSA-Gd produced obviously enhanced cell lethality compared with ICG and Au@BSA-Gd group, which demonstrated the improved phototoxicity. Fluorescence staining studies using calcein AM and PI to stain different formulations treated 4T1 cells were performed for visualization of the live (green) and dead cells (red). As indicated in Figure 5d, 4T1 cells treated with NIR laser alone exhibited vivid green, suggesting the safety of irradiation in such conditions. However, the

cell

viability

and

density

decreased

to

different

extents

in

other

formulations-treated groups. Noticeably, the optimal formulation (ICG-Au@BSA-Gd) compromised the largest amounts of cell apoptosis and necrosis by a laser-activated PTT/PDT synergistic effect. Additionally, the FITC-Annexin V/PI double staining flow cytometry results (Figure 5e), investigated the enhanced therapeutic efficiency by simultaneous PTT



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28

/PDT effect of the optimal formulation, which was consistent well with the former MTT and co-stained data. ICG-Au@BSA-Gd treated group exhibited significantly more late apoptosis and necrosis by combined PTT/PDT effect, compared with ICG or Au@BSA-Gd group. Overall, all results evidently pointed the superior of ICG-Au@BSA-Gd nanocomposites for their 808 nm light-drived combined PTT/PDT therapeutic efficiency. 3.9. In Vivo NIRF/PA/CT/MR Quad-Modal Imaging. The ideal nano-agent should be efficiently accumulated in tumor sites and detected for accurate cancer therapy. On the basis of the in vitro imaging treatment results, we further applied the ICG-Au@BSA-Gd to mice for NIRF/PA/CT/MR quad-modal imaging. First, we monitored time-dependent biodistribution of the ICG-Au@BSA-Gd nanoparticles (1.0 mg ICG/kg) using in vivo NIR fluorescence microscopy after intravenous administration (Figure 6a). The NIRF signals of free ICG group 1 h after injection were extensive in the liver rather than in the tumor. With the signals gradually diminished as time passed, it nearly disappeared at 24 h post-injection. On the contrary, weak fluorescence signals were detected at tumor sites 1 h after ICG-Au@BSA-Gd injection, then gradually increased and reached the highest level at 12 h. It was important to note that the strong fluorescence intensity in the tumor still remained steady at 24 h, implying that the formulations could successfully reach at tumors by EPR effect and stay for a prolonged period of time. Ex vivo NIRF imaging was also performed to further measure the ICG-Au@BSA-Gd distribution. Tumors and major organs (heart, liver, spleen, lung, and kidneys) were excised after 24 h.


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Evidently, most ICG in ICG-Au@BSA-Gd assembled in the tumor sites instead of the liver or other metabolic organs, compared with a dramatic liver accumulation of free ICG, which was corresponding to semi-quantitative NIRF intensity detection (Figure 6b). Additionally, we assumed the in vivo fluorescence signal were from the released ICG because of the quenched fluorescence of loaded ICG (Figure S2). To verify this assumption, we measured the in vitro fluorescence signals of the released ICG at different time points. As displayed in the Figure S6, we could see that the fluorescence signals of the ICG increased over the time, indicating the release of loaded photosensitizer and its regained fluorescence, which was in favor of achieving fluorescence imaging-guided phototherapy for the quenched ICG-Au@BSA-Gd formulations. The in vivo PA imaging ability was further evaluated, which integrated the advantages of deep tissue penetration and fine sensitivity. PA imaging was performed before and after intravenous injection of the Au@BSA-Gd and ICG-Au@BSA-Gd solutions into tumor-bearing mice. The PA signals of both Au@BSA-Gd and ICG-Au@BSA-Gd gradually increased over time in the tumor regions and achieved the maximum at 12 h post-injection (Figure S7, 6c). Quantitative analysis in Figure 6d showed the PA signal intensity of the ICG-Au@BSA-Gd at 12 h was enhanced by 8.8 fold compared with the preinjected signal, suggesting the effective tumor homing of the final formulations, which consisted well with the FL imaging results. Gold as a high atomic number element could cause a strong photoelectron effect to X-ray attenuation.42 We deduced the ICG-Au@BSA-Gd could also provide



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30

the ability for CT imaging in vivo, besides the ideal effect as photothermal agent. 4T1 tumor-bearing mice were injected with the ICG-Au@BSA-Gd (25 mg/kg) nanocomposites for examining the CT contrast performance. A significant contrast increase was observed at the tumor site after 12 h intravenous injection (Figure 6e). Additionally, the CT numbers of hounsfield units (HU) of the whole tumor area had a 2.3-fold increase from 125 to 286 HU (Figure 6f). With BSA-Gd hybrid included in the system, T1-weight MR images were also recorded before and after ICG-Au@BSA-Gd (0.08 mmol Gd/kg) injection using a 7T MR instrument. The tumor area after 12 h intravenous injection displayed a much brighter image with a sharp border than the preinjection images (Figure 6g), representing a 457.1% raising of T1-weighted signal intensity (Figure 6h). These results confirmed ICG-Au@BSA-Gd nanoparticles could act as an excellent contrast agent for NIRF/PA/CT/MR quad-modal in vivo imaging by combing different imaging modalities in one therapeutic schedule, thus triumphantly providing more precise tumor information from a broad different spectrum of intrinsic optical, electronic, and magnetic perspective. The diagnostic imaging platform also supplied improved feasibility and efficacy, and thus stimulated considerable interest in the biomedical field toward imaging with higher sensitivity and resolution. 3.10. In Vivo Blood Circulation and Biodistribution. In vivo blood circulation and biodistribution of ICG-Au@BSA-Gd nanoparticles were further evaluated after intravenous injection, to verify the above imaging experiments. In blood circulation behavior studies, blood samples were collected intermittently at indicated time points.


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Au3+ levels in the blood samples were detected using inductively coupled plasma mass spectrometry (ICP-MS). As seen in the blood circulation curve (Figure 7a), ICG-Au@BSA-Gd exhibited an enduring exist and the half-time of blood circulation could reach 3.02 ± 0.33 h. After administrated with the final formulation for 24 h, we tested the in vivo biodistribution. In Figure 7b, reticuloendothelial systems (RES) such as liver and spleen showed the highest uptake of ICG-Au@BSA-Gd. Besides, the tumor tissues also exhibited relative high uptake due to the effective EPR effect and longer circulation time, which was consistent well with the former quad-modal imaging data. Moreover, no noticeable distinction of Au and Gd amount was detected, verifying the ideal stability of ICG-Au@BSA-Gd. Therefore, the passively targeted ICG-Au@BSA-Gd is capable of displaying the tumor sites by multimodal imaging to guide the phototherapy. 3.11. In Vivo Synergistic Antitumor Efficacy of ICG-Au@BSA-Gd Nanoparticles. All of the imaging results indicated that ICG-Au@BSA-Gd largely assembled at the tumor sites after 12 h intravenous injection. To investigate the antitumor in vivo PTT/PDT efficacy, the photothermal effect of ICG-Au@BSA-Gd was first monitored by an infrared thermal imaging camera (Figure 7c). Temperature changes as a function of irradiation time were displayed in Figure 7d. After the tumor region was exposed to a laser irradiation 12 h post-injection, the central temperature of free ICG, Au@BSA-Gd and ICG-Au@BSA-Gd treated groups all presented a distinct temperature elevation with increasing irradiation time. Besides, the synergistic formulation group reached uppermost temperature to 56.8 °C after 5 min



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32

irradiation, exceeding the damage threshold to induce irreversible tissue damage. As the results indicated, the ICG-Au@BSA-Gd has effectively caused photo-heat transfer for photothermal cancer therapy. The ROS production at the tumor site is also a vital index of PDT efficacy in vivo. It was obvious that no fluorescence of ROS was found in PBS and ICG-Au@BSA-Gd treated group. What’s more, the histopathologic image of ICG-Au@BSA-Gd without laser-treated tumor exhibited the intratumoral distribution. Noticeably, evident levels of green DCFH-DA (ROS) fluorescence were observed in ICG-Au@BSA-Gd plus laser group, while free ICG with light exposure induced a relatively lower one (Figure 8e), which was attributable to the effective accumulation of ICG-Au@BSA-Gd in the tumors. After phototherapy, the tumor volume and body weight of mice were monitored every 3 day for 21 days. As indicated in Figure 8b, tumor growth was relatively rapid in both PBS and NIR groups, indicating that simple thermal therapy with current laser treatment was ineffective at inhibiting tumor growth. ICG or Au@BSA-Gd coupled with laser irradiation hindered tumor growth to a certain extent, demonstrating the valid therapeutic efficacy of single ICG or Au@BSA-Gd respectively. However, synergistic effect of ICG-Au@BSA-Gd displayed a strongest inhibition to tumor growth when subjected to laser exposure. We further captured the tumor changes of different formulation treated group during the treatment. Figure S8 showed the tumors of ICG-Au@BSA-Gd group was largely decreased and left the original tumor sites with black scars compared with the control group. At the end of the 21 days


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treatments, the mice were executed and tumors were isolated and further measured. The photos of isolated tumors from representative groups (Figure 8a) displayed the tumor size of optimal therapy (ICG-Au@BSA-Gd + 808 nm light) was minimum, verifying the excellent tumor inhibition effect. What’s more, the body weight of each treatment group did not apparently change (Figure 8c), suggesting the lower systemic toxicity. Moreover, the survival curves of each group were also recorded in another treated experiment except for the longer performing days. The results demonstrated treatment of ICG-Au@BSA-Gd combined with 808 nm laser was excellent in prolonging the mice survival (an 80% survival after 35 days, Figure 8d) The harvested tumors and organs were then subjected to histopathological analysis according to hematoxylin and eosin (H&E) staining (Figure 8f). The structure of residual tumor tissues of the ICG-Au@BSA-Gd plus NIR group suffered more apparent extensive tumor damage, such as nuclear membrane fragmentation and nuclei shrinkage with pyknosis (condensation of chromatin).43 Noticeably, the optimal group exhibited less obvious necrosis and inflammation of the major organs, compared with the control group, validating the insignificant toxic side effects of ICG-Au@BSA-Gd. Thus, considering the ideal theranostic capability without significant cytotoxicity, ICG-Au@BSA-Gd integrated with 808 nm laser, was chosen as an ideal candidate for light triggered PTT/PDT in vivo.

4. CONCLUSION In this work, albumin-bioinspired gadolinium hybrids functionalized hollow gold nanoshells

(Au@BSA-Gd)

were

successfully

designed.


The

fabricated


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nanocomposites exhibited pronounced PA/CT imaging and T1-weighted MR signals as well as a strong NIR photothermal conversion capability with remarkable water-dispersiblity and biocompatiblity. Meanwhile, Au@BSA-Gd nanoplatform showed high loading capacity for the therapeutic agent indocyanine green (ICG) owing to the hollow porous gold nanoshells and conjugated serum albumin. After loading ICG, the Au@BSA-Gd nanocomposites demonstrated excellent in vivo quad-modal NIRF/PA/CT/MR imaging capability, as well as combined photothermal and photodynamic therapy. Overall, the as-prepared ICG-Au@BSA-Gd based nanoplatform highlighted the great potential as theranostic agent for precise diagnosis and therapy of cancer.

ASSOCIATED CONTENT Supporting Information. Zeta potential, fluorescence spectra, stability, CLSM images of 4T1 cells treated with ICG-Au@BSA-Gd, in vitro fluorescence signals of released ICG, in vivo PA images of Au@BSA-Gd and representative photographs of treated mice.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (81503016),

National

Basic

Research

Project (973

Program)

of

(2014CB932200), and Peiyang Young Talent Fund of Tianjin University (1701)


China

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Figures:

Figure 1. Schematic illustration of the ICG-Au@BSA-Gd theranostic agents for NIRF/PA/CT/MR quad-modal imaging guided simultaneous PTT/PDT effect for cancer therapy.



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Figure 2. Preparation and characterization of the hollow gold nanoshells (HAuNs). (a) Preparation method of the HAuNs. (b) TEM images of mSiO2 (I), mSiO2@Au seeds (II), procedure of gold seeds growth of the mSiO2@Au seeds nanocomposites (III and IV), mSiO2@Au nanoshells (V) and HAuNs (VI), respectively. (c) UV-visible spectra of the mSiO2 (curve 1), mSiO2@Au seeds (curve 2); gold seeds growth curves (curve 3-5); final formation of mSiO2@Au nanoshells (curve 6) and HAuNs (curve 7). (d) The digital photograph of the different formulations during the different stages of gold seeds growth process corresponding to the UV-visible spectra. (e) EDX mapping images of HAuNs after removal of the inner silicon. (f) Scanning electron microscopy (SEM) images of mSiO2 (I, II) and HAuNs (III, IV).


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Figure 3. (a) Synthesis process of the ICG-Au@BSA-Gd formulations. (b) TEM image of the ICG-Au@BSA-Gd nanoparticles. (c) Size distribution of HAuNS and Au@BSA-Gd by dynamic light scatterings. (d) The FTIR spectra of different formulations in the process of the formation of Au@BSA-Gd. (e) CD spectra of pure BSA, BSA-Gd and Au@BSA-Gd, respectively. (f) Photographs of different formulations before and after loading ICG into the Au@BSA-Gd (1-4 represents BSA-Gd, Au@BSA-Gd, ICG and ICG-Au@BSA-Gd, respectively). (g) UV-visible spectra of BSA-Gd, Au@BSA-Gd, free ICG, ICG/Au@BSA-Gd and ICG-Au@BSA-Gd, respectively. (h) The loading capacity based on the different ICG concentration (For Au@BSA-Gd 1.0 mg and ICG solution 1mL). (i) The release behaviors of ICG from ICG-Au@BSA-Gd in different pH values of PBS.



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Figure 4. In vitro PA/CT/MR imaging and evaluation of PTT/PDT effect. (a) PA imaging phantoms, consisting of various concentrations of ICG loaded in the Au@BSA-Gd nanoparticles. Lower is the linear relationship between PA signal intensity and different ICG concentration. (b) CT images and corresponding values of the ICG-Au@BSA-Gd nanoparticles at the indicated Au concentrations. Lower is the CT values of the ICG-Au@BSA-Gd aqueous dispersions at the determined Au concentrations. (c) T1-weighted MR images of ICG-Au@BSA-Gd at varying Gd3+ concentrations. Lower is the linear relationship between T1 relaxation rate (1/T1) and Gd3+ concentrations in ICG-Au@BSA-Gd aqueous solutions. (d) NIR-triggered temperature elevations of Au@BSA-Gd nanoparticles with different Au concentrations over a period of 5 min laser irradiation (808 nm, 1.5 W/cm2). (e) NIR-triggered temperature elevations of different curves of PBS, Au@BSA-Gd (100 µg/mL), ICG (10 µg/mL) and ICG-Au@BSA-Gd (10 µg/mL for ICG and 100 µg/mL for Au@BSA-Gd) over a period of 5 min laser irradiation (808 nm, 1.5 W/cm2). (f) (g) Photothermal effect of the irradiation of the aqueous solution of the ICG-Au@BSA-Gd nanoparticles (10 µg/mL for ICG and 100 µg/mL for Au@BSA-Gd) with the NIR laser irradiation (808 nm, 1.5 W/cm2), where the laser was first irradiated for 600 s and then removed. (h) Linear time data versus -ln (θ) obtained from the cooling period of Fig.4g. (i) Changes of the absorbance of DPBF at 410 nm in different formulations as a function of 808 nm laser irradiation. (j) Thermographic images of various solutions at determined time intervals (2.5 min and 5 min, respectively).


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Figure 5. Cell viability of 4T1 cells incubated with various concentrations of free ICG, Au@BSA-Gd, and ICG-Au@BSA-Gd without (a) or with (b) NIR laser irradiation (808 nm, 1.5 W/cm2, 5 min). Data are presented as means ± SD (n = 3). (c) CLSM images of 4T1 cells treated with PBS, free ICG + NIR, ICG-Au@BSA-Gd and ICG-Au@BSA-Gd + NIR for ROS detection. (NIR means the cells were exposed to 1.5 W/cm2, 808 nm laser for 5 min). Scale bar: 25 µm. (d) CLSM images of 4T1 cells treated with PBS, free ICG, Au@BSA-Gd, and ICG-Au@BSA-Gd with NIR laser irradiation (808 nm, 1.5W/cm2, 5 min). Live cells were stained green with calcein-AM, and dead/later apoptosis cells were stained red with PI. Scale bar: 200 µm. (e) Flow cytometry results of FITC-Annexin V/PI double stained 4T1 cells treated with PBS (I), free ICG (II), Au@BSA-Gd (III) and ICG-Au@BSA-Gd (IV), followed by laser irradiation (808 nm, 1.5 W/ cm2, 5 min ).



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Figure 6. In vivo NIRF/PA/CT/MR imagings. (a) Fluorescence images of tumor bearing mice at different time points after administration of free ICG and ICG-Au@BSA-Gd nanoparticles; the bottom panel shows the ex vivo images examined at 24 h post-injection. (b) Relative fluorescence intensity of ICG in major organs after administration with free ICG and ICG-Au@BSA-Gd nanoparticles for 24 h, (**) P < 0.01. (c) PA images of 4T1 tumor-bearing mice after intravenously injected ICG-Au@BSA-Gd nanoparticles at different time points. (d) Corresponding photoacoustic signal intensity of ICG-Au@BSA-Gd nanoparticles in the tumor at the corresponding time points. (e) In vivo CT images (Left for the 3D and right for the 2D, respectively) of mice before (pre) and after 12 h injection with ICG-Au@BSA-Gd. (f) Corresponding HU value of ICG-Au@BSA-Gd nanocomposites in the tumor sites before and 12 h injection, (**) P < 0.01. (g) T1-weighted MR images before (pre) and after 12 h treatment with ICG-Au@BSA-Gd NPs. (h) The normalized MR signal intensity in the tumor sites before (pre) and after 12 h injection, (**) P < 0.01.


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Figure 7. (a) Pharmacokinetic profiles of ICG-Au@BSA-Gd nanoparticles following intravenous administration by measuring Au concentrations. (b) The bio-distribution of Au and Gd in tumor sites and main tissues after 24 h of intravenous administration of ICG-Au@BSA-Gd nanoparticles. (c) Infrared thermographic images of tumor-bearing mice exposed to an 808 nm laser (1.5 W/cm2, 5 min) for determined time intervals 12 h post intravenous injection of different formulations. (d) Tumor temperature rise curves of mice injected with different formulations as a function of irradiation time.



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Figure 8. (a) Photographs of tumor tissues from different groups after 21 day treatments. (b) Tumor growth in tumor bearing mice after various treatments and laser irradiation, (*) P < 0.05, (**) P < 0.01. (c) Body weight changes of mice bearing tumors as a function of days post treatments for various groups (n =10). (d) Percent survival of different treatment groups (n =10). (e) Representative intratumoral DCFH-DA fluorescence images as an indicator of ROS generation level. Scale bar: 100 µm. (f) H&E stained images of major organs gathered from PBS and ICG-Au@BSA-Gd+NIR groups after continuous treatments for 21 days. Scale bar: 25 um.


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TOC Graphic 234x149mm (150 x 150 DPI)


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