Indocyanine Green Loaded Magnetic Carbon Nanoparticles for Near

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Indocyanine Green Loaded Magnetic Carbon Nanoparticles for Near Infrared Fluorescence/Magnetic Resonance DualModal Imaging and Photothermal Therapy of Tumor Saijie Song, He Shen, Tao Yang, Lina Wang, Han Fu, Huabing Chen, and Zhijun Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00490 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 4, 2017

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ACS Applied Materials & Interfaces

Indocyanine Green Loaded Magnetic Carbon Nanoparticles for Near Infrared Fluorescence/Magnetic Resonance Dual-Modal Imaging and Photothermal Therapy of Tumor Saijie Song, Zhang b,*

a,b

He Shen, b,* Tao Yang,

c

Lina Wang, b Fu Han,

b

Huabing Chen,

c,d

Zhijun

a

School of Nano Technology and Nano Bionics, University of Science and Technology of China, Hefei 230026, China

b

CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine, CAS Center for Excellence in Nanoscience, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China

c

Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China d

School for Radiological & Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China

*Corresponding authors: E-mail addresses: [email protected] (H. Shen) [email protected] (Z. Zhang)

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ACS Paragon Plus Environment


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Abstract: Malignant tumor incidences have been rapidly rising recently and are becoming a serious threat to human health. Herein, a multifunctional cancer targeted theranostic nanoplatform is developed by in situ growth of iron oxide magnetic nanoparticles on carbon nanoparticles, and then loaded with fluorescent dye indocyanine green (ICG@MCNPs). The loading of ICG on the nanoplatform significantly improves its photostability, and hence facilitates long-term near infrared fluorescence (NIRF) imaging and efficient photothermal therapy (PTT) of tumor. The in vivo NIRF imaging reveals that ICG@MCNPs can be targeted to the tumor site. Moreover, in vivo magnetic resonance imaging also confirmed the efficient accumulation of ICG@MCNPs in the tumor site. Inspiringly, the subsequent PTT of tumor-bearing mice is achieved, as evidenced by the complete ablation of the tumor and the recovery of the physiological indexes to normal levels. Benefited from its low-cost, simple preparation, and excellent dual-modal imaging and therapy, the ICG@MCNPs-based theranostic nanoplatform holds great promise in tumor-targeted nanomedicine.

Keywords: indocyanine green; magnetic carbon nanoparticles; theranostic nanoplatform; dual-modal imaging; photothermal therapy.

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1. Introduction Cancer has ranked among one of the most severe global health issues.1 According to the World Cancer Report 2014 compiled by the International Agency for Research on Cancer (IARC) of World Health Organization (WHO), ~14.1 million new cases of cancer have raised worldwide in 2014, and the global anti-cancer challenge would be more severe in the next two decades.2 To defeat this very stubborn disease, various diagnostic tools, such as magnetic resonance imaging (MRI), near-infrared fluorescence (NIRF) imaging, computed tomography (CT), and therapeutical approaches, such as surgery, chemotherapy, radiotherapy, photothermal therapy (PTT), photodynamic therapy (PDT) and immunotherapy, have been developed over the past several decades.3-6 However, the traditional diagnostic or therapeutical agents, including small organic molecules and chemical drugs, are often limited by many factors, such as nonspecific targeting and short circulation time, impeding the pace of efficient personalized therapy.7 Recently, nanotechnology and nanomaterials, which possess excellent passive targeting ability to tumor site via enhanced permeability and retention (EPR) effect and long blood circulation time, have provided more chances to defeat cancer efficiently.8-11 Various nanomaterials, such as polymeric nanocarriers, quantum dots, noble metal nanoparticles, magnetic nanoparticles and carbonaceous nanomaterials, have been developed as theranostic agents for simultaneous cancer imaging and therapy.12-16 Among various nanotheranostic agents, NIRF dyes have attracted much interest for their excellent in vivo deep penetrating NIRF imaging and photothermal effects.17-20 Indocyanine green (ICG), an NIRF dye approved by U. S. Food and Drug Administration (FDA) as a clinical imaging agent, has also proved to be a promising clinical translational agent for effective and safe PTT.21-23 3



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However, the poor photostability, nonspecific targeting, and rapid in vivo elimination hamper its clinical application.24 Hence, multifarious vectors, including protein cages,25, 26 lipid polymers,27 and graphene oxide (GO),28 have been developed to overcome the limitations of free ICG. Among various vectors developed, GO has attracted significant attention for its ultrahigh adsorption capacity and drug loading capability, excellent photostability, enhanced PTT effect, and passive tumor targeting by EPR effect.29-31 For instance, Hu et al. developed a theranostic nanoplatform based on ICG loaded polydopamine-reduced GO (ICG-PDA-rGO) for enhanced PTT of cancer.28 Similarly, Sharker et al. also reported the enhanced tumor-targeting and PTT effect of ICG integrated with rGO.31 Although the ICG loaded GO showed better PTT performance, the large-sized GO was preferentially accumulated in perivascular regions, and hindered its diffusion in tumor site.9 Besides, large-sized GO tends to aggregate in some organs (e. g. liver, kidney and spleen), generating potential long-term toxicity.33, 34 In regards to the size-dependent potential toxicity, our previous work also revealed that GO with small size exhibited much lower hazard than that with jumbo size.33 Recently, we have developed small-sized carbon nanoparticles (CNPs) (with size less than 10 nm) for drug delivery and theranostic applications.35, 36 CNPs and CNPs composites possess excellent physicochemical stability, good biocompatibility, and outstanding drug loading capacity, and therefore are promising nanovectors for ICG delivery. Fluorescence imaging is of high sensitivity, however its poor tissue penetration and spatial resolution limited its clinical application. To remedy the defect of fluorescence imaging, the combination of MRI, which exhibits high temporal-spatial resolution and tissue penetration, is introduced. As previously reported, the hybrid nanomaterials, by in situ growth of magnetic nanoparticles on carbonaceous namomaterials, exhibited enhanced MRI effect.37, 38 For instance, 4


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Liu group reported superparamagnetic GO-iron oxide for magnetically targeted drug delivery and MRI guided PTT of tumor.39 Our recent work indicated that iron oxide nanoparticles (IONPs) supported by graphene quantum dots possessed high transverse relaxivity (r2=134.3 mM-1s-1) and excellent in vivo MRI effect.37 In consideration of the enhanced MRI effect of carbonaceous nanomaterials loaded with magnetic nanoparticles, we expect that magnetic CNPs (MCNPs) should possess satisfactory MRI performance for tumor diagnosis.35-37 Based on the above discussion, we herein report rational design and development of ICG loaded MCNPs for NIRF/MR dual-modal imaging and PTT of tumor: In our strategy, CNPs was prepared by oxidation of activated carbon, and onto which iron oxide nanoparticles was subsequently grown in situ, to form MCNPs. ICG was then loaded onto the MCNPs via physisorption. Bovine serum albumin (BSA) was finally coated on the MCNPs to improve their physiological stability and biocompatibility (Scheme 1). The adsorption of ICG to the MCNPs, we demonstrated, leads to excellent ICG loading performance, increased photostability and consequent enhanced photothermal effect. In vitro and in vivo experiments indicated that the ICG@MCNPs showed efficient accumulation and long retention behavior in tumor site, and finally resulted in excellent NIRF/MR imaging and enhanced PTT of tumor.

2. Experiment Section 2.1. Materials Indocyanine

green

(ICG)

was

purchased

from

Sagon

Biotech.

3-(4,

5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma. Other reagents were purchased from Sinopharm Chemical Reagent Company.

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2.2. Instrumentation Atomic force microscopy (AFM) imaging was performed with a Vecco Dimension 3100 atomic force microscope. UV-Vis-NIR spectra were collected in the 200~900 nm wavelength range with a Perkin-Elmer Lambda 25 spectrophotometer. Transmission electron microscopy (TEM) imaging and high-resolution transmission electron microscopy (HRTEM) imaging were collected at an accelerating voltage of 200 kV (Tecnai G2 F20 S-Twin TEM). Dynamic light scattering (DLS) analysis was conducted by a particle size/zeta analyzer (ZEN3600-nanoZS, Malvern). Hysteresis loop was measured on a Quantum Design physical property measurement system (PPMS-9T, EC-II). X-ray diffraction (XRD) spectra was obtained by using a Bruker D8 ADVANCE diffractometer (Germany) with Cu Kα (λ=1.5406Å). The cell imaging was obtained on a Nikon A1 confocal laser scanning microscope.

2.3. Synthesis of ICG@MCNPs CNPs were prepared via chemical oxidation as described in our previous report with minor modification. Briefly, 2 g of activated carbon was added to a mixed solution of 30 mL fuming HNO3 and 90 mL concentrated H2SO4 in an ice bath under mild magnetic stirring for 5 min. Then the reaction mixture was kept at 120 oC for 8 min under vigorous stirring in an oil bath. The acquired black-yellow suspension was then cooled down immediately in an ice bath and slowly diluted with 600 mL D. I. water. The acidic solution was neutralized to pH 7-8 with NaOH and Na2CO3; the resulting solution was further dialyzed for 2 d (100 kDa cutoff) to remove inorganic salts and small sized CNPs. Finally, the solution was concentrated on a rotary evaporator and further purified via spin dialysis (100 kDa, 6000 rpm). 6


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MCNPs were obtained via a one-step co-precipitation method, in which process iron oxide grew on the CNPs surface in situ. Next, 1 g CNPs were added into a 2 L glass beaker containing 1 L D. I. water under vigorous stirring at 80 oC, followed by adding 5 g iron dichloride tetrahydrate and 7.5 mL 25% ammonium hydroxide. After 4 h, the solution was cooled down to room temperature and then dialyzed for 2 d (100 kDa cut-off). The acquired MCNPs were concentrated via spin dialysis (100kDa, 6000 rpm) for further use. IONPs were obtained via a similar process. Briefly, 1 g sodium citrate was added into a 2 L glass beaker containing 1 L D. I. water under vigorous stirring at 80 oC, followed by adding 5 g iron dichloride tetrahydrate and 7.5 mL 25% ammonium hydroxide. After 4 h, the solution was cooled down to room temperature and then dialyzed for 2 d (100 kDa cut-off). The acquired IONPs were concentrated via spin dialysis (100 kDa, 6000 rpm) for further use. ICG@MCNPs were formed by a mild physisorption approach. First, 1 g ICG and 4 g BSA were added into 2 L aqueous solution of MCNPs (calculated from 0.5 mg/mL of CNPs) under mild stirring overnight at room temperature in the dark. Free ICG and BSA were removed by spin dialysis (100 kDa, 6000 rpm, 10 min) and the acquired green-brown solution was stored for further use. The amount of [Fe] was determined by ICP-OES (VARIAN, 710-ES), and the ICG loading capacity was calculated by the following equation: Loading efficiency=WICG/WMCNPs×100% Where WICG is the weight of ICG loaded on MCNPs, and WMCNPs is the weight of MCNPs.

2.4. Cell culture 4T1 cells (Shanghai Institutes for Biological Science, Chinese Academy of Sciences) were 7



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cultured in regular Roswell Park Memorial Institute (RPMI) 1640 medium (Corning) containing 10% fetal bovine serum (FBS, Hyclone), 100 U/mL penicillin and 100 U/mL streptomycin in a humidified atmosphere of 37 oC and 5% CO2.

2.5. Cell viability assay MTT assay was employed to evaluate the cytotoxicity of ICG@MCNPs. 4T1 cells were seeded into 96-well plates at a density of 1×104 cells per well (with 200 µL culture medium), and maintained for 24 h (5 % CO2, 37 oC). Then ICG and ICG@MCNPs were introduced into cells with various final concentrations (1, 3, 6, 12, 25 and 50 µg/mL, respectively, calculated according to ICG concentration) and incubated for another 24 h. Then the medium was discarded and the cells were washed three times using PBS. The cells treated with PBS were set as the control (100%). Following this procedure, the relative cell viability was evaluated via the standard MTT assay.

2.6. Cellular uptake of ICG@MCNPs 5×104 4T1 cells were seeded into each well of 35 mm chambered coverglass and cultured for 12 h. The old medium was replaced with medium containing ICG@MCNPs (10 µg/mL of ICG). After 6, 12, and 24 h incubation, the medium was removed and the cells were washed with PBS for 3 times. Then, the cells were fixed with 4% paraformaldehyde solution for 20 min, stained with DAPI for 10 min, and rinsed three times with PBS. Cellular uptake was observed on a Nikon A1 confocal laser scanning microscope. For semi-quantitative analysis of the uptake of ICG@MCNPs, 2×105 4T1 cells were seeded 8


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into each well of 6-well plates and maintained for 12 h (5 % CO2, 37 oC). After the old medium was replaced with fresh medium, ICG@MCNPs (with 10 mg/L ICG) was added into each well at various time points (0.1, 0.5, 2, 6, 12, and 24 h). The cells were washed with PBS for 3 times, digested, and resuspended in PBS. At last, each group of the cells was added into a 96-well black plate (2×104 cells with 0.1 mL PBS), and the fluorescence images were recorded by a Cri maestro ex in vivo imaging system (USA).

2.7. In vitro photothermal effects and toxicity of ICG@MCNPs To evaluate the photothermal effect of ICG@MCNPs, 0.5 mL aqueous solutions at various ICG concentrations (1-50 µg/mL) were irradiated under an NIR laser irradiation (808 nm, 2 W/cm2) for 5 min. During the irradiation process, the temperature of the solutions were recorded every 30 s by a digital thermometer. To evaluate the photothermal toxicity, 4T1 cells were first seeded into 96-well plates at a cell density of 104/well. After 24 h, old media were replaced by 100 µL fresh media containing PBS (control), free ICG and ICG@MCNPs (ICG concentration: 1, 3, 6, 12, 25 and 50 µg/mL). After another 24 h, the plate was placed on a digital dry bath incubator to keep it at 37 oC. The cells were irradiated with/without an 808 nm laser at 2 W/cm2 for 3 min. The cell viability was evaluated via MTT assay. The results represent average values (n=3).

2.8. Animals and tumor model Healthy female BaLb/c mice at an average age of 6-7 weeks were purchased from Suzhou Industrial Park Animal Technology Co., Itd. All animals received care in accordance with the 9



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Guidance Suggestion for the Care and Use of Laboratory Animals. The procedures were approved by Soochow University Laboratory Animal Center. 4T1 cells (2×106 cells in 50 µL serum free cell culture medium) were subcutaneously injected into the 6-7 week old mice at the right hind leg. The tumor volume was calculated according to the following equation: (Tumor length) ×(tumor width) 2/2

2.9. MR imaging T2 relaxation time of MCNPs at different concentrations was measured via an 11.7 T MRI scanner under the following measurement parameters: repetition time (TR)=5000 ms, echo time (TE)=8.0 ms, imaging matrix=128×128, slice thickness=0.8 mm, field of view (fov)= 1.5×1.5 cm, and number averages (NA)=2. For in vivo MRI, 100 µL aqueous solution of ICG@MCNPs was injected intravenously into tumor-bearing mice ([Fe]=5 mg/kg) via tail vein. MR images were obtained at 0 and 6 h p. i. The measurement parameters included the following: TR=3000 ms, TE=22.0 ms, imaging matrix=156×156, slice thickness=0.8 mm, fov= 2.5×2.5 cm, and NA=4.

2.10. NIRF imaging For in vivo NIRF imaging, aqueous solutions of ICG and ICG@MCNPs (5 mg/kg of ICG) were injected intravenously into tumor-bearing BaLb/c nude mice (female, 6-7 week) via tail vein when the tumor volumes grew up to ~50 mm3. The Cri maestro ex in vivo imaging system (USA) was employed to obtain the fluorescence signals of ICG (704 nm excitation and 735 nm filter) at 0, 10


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1, 2, 4, 6, 8, 10, 24 and 36 h p. i. The fluorescence images of various organs (heart, liver, spleen, lung, kidney) and tumor at 2, 6, 12, and 36 h p. i., and the blood samples at 0, 0.5, 2, 4, 6, 10, 24, and 36 h p. i. were collected by the Cri maestro ex in vivo imaging system (USA).

2.11. Thermal imaging analysis 100 µL of PBS and ICG/MCNPs (5mg/kg of ICG) were injected intravenously into tumor-bearing BaLb/c mice (female, 6-7 week) via tail vein when the tumor volumes grew up to ~50 mm3. After 6 h p. i., an 808 nm laser at 2 W/cm2 was employed to irradiate the tumor sites for 5 min. An infrared thermal imaging camera was used to record infrared thermographic maps of the tumor and region temperatures during the NIR laser irradiation process.

2.12. In vivo photothermal therapy 25 healthy female BaLb/c mice (6-7 weeks) were divided into 5 groups randomly: normal mice group (control, no tumor-bearing), PBS group, ICG@MCNPs group, PBS + IR group and ICG@MCNPs + IR group. The 4T1 tumor-bearing mice were randomly separated into four groups of five mice. Once the average tumor size reached 50 mm3, 100 µL PBS and ICG@MCNPs (1mg/mL) was intravenously injected into the mice via tail vein at a single dose of 5 mg/kg of ICG. Then, the mice were anaesthetized with 100 µL 10% chloral hydrate and with/without suffered from NIR laser irradiation (808 nm, 2W/cm2) for 5 min at 6 h p. i. The size of the tumor and the weight of the mice tested were recorded every other day during the following 3 weeks to evaluate the in vivo PTT efficiency. All of the mice were sacrificed by cervical dislocation under anesthesia after experiments. 11



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2.13. Blood biochemical analysis and histopathological assessment About 0.8 mL of blood was collected from each mouse and used for further blood chemistry tests and complete blood panel analysis before the mice were euthanatized. Major organs (including the heart, liver, spleen, kidney and lung) and tumors were harvested and fixed in 4% neutral buffered formalin. After tissue sectioning and H & E staining, micromorphology of tumors and tissues were recorded under a digital microscope.

2.14. Statistical analysis Statistics were based on standard deviation of 5 mice per group. One-tailed Student’s test was applied to evaluate the significance among groups, and p

Indocyanine Green Loaded Magnetic Carbon Nanoparticles for Near

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