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Improving the anticancer efficacy of laminin receptor-specific therapeutic ruthenium nanoparticles (RuBB-loaded EGCGRuNPs) via ROS-dependent apoptosis in SMMC-7721 cells Yanhui Zhou, Qianqian Yu, Xiuying Qin, Dhairya Bhavsar, Licong Yang, Qingchang Chen, Wenjing Zheng, Lanmei Chen, and Jie Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b02261 • Publication Date (Web): 27 May 2015 Downloaded from http://pubs.acs.org on June 1, 2015

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ACS Applied Materials & Interfaces

Improving the anticancer efficacy of laminin receptor-specific therapeutic ruthenium nanoparticles (RuBB-loaded EGCG-RuNPs) via ROS-dependent apoptosis in SMMC-7721 cells Yanhui Zhou 1, Qianqian Yu1, Xiuying Qin1, Dhairya Bhavsar1, Licong Yang1, Qingchang Chen1, Wenjing Zheng1 Lanmei Chen1 and Jie Liu 1* 1 Department of Chemistry, Jinan University, Guangzhou 510632, China

Keywords: ruthenium nanoparticles, cellular targeting, 67LR receptors, reactive oxygen species, fluorescence imaging

* Corresponding author: Address: Department of Chemistry, Jinan University, Guangzhou 510632, China; Tel.: +86 20 85220223; fax:85220223 E-mail addresses: [email protected] (J. Liu). 1

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ABSTRACT

Functionalization can promote the uptake of nanoparticles into cancer cells via receptor-mediated endocytosis, enabling them to exert their therapeutic effects. In this paper, epigallocatechin gallate (EGCG), which has a high binding affinity to 67-kDa laminin receptor overexpressed in HCC cells, was employed in the present study to functionalized ruthenium nanoparticles (RuNPs) loaded with luminescent ruthenium complexes to achieve anti-liver cancer efficacy. RuBB-loaded EGCG-RuNPs showed small particle size with narrow distribution, better stability, and high selectivity between liver cancer and normal cells. The internalization of RuBB-loaded EGCG-RuNPs was inhibited by 67LR-blocking antibody or laminin, suggesting that 67LR-mediated endocytosis played an important role in the uptake into HCC cells. Moreover, TEM and confocal microscopic images showed that RuBB-loaded EGCG-RuNPs accumulated in the cytoplasm of SMMC-7721 cells. Furthermore, our results indicated that the EGCG-functionalized nanoparticles displayed enhanced anti-cancer effects on a target specific manner. Concentrations of RuBB-loaded EGCG-RuNPs, nontoxic in normal L-02 cells, showed direct reactive oxygen species-dependent cytotoxic, pro-apoptotic, and anti-invasive effects in SMMC-7721 cells. Furthermore, in vivo animal study demonstrated that RuBB-loaded EGCG-RuNPs possessed high anti-tumor efficacy on tumor-bearing nude mice. It is encouraging to conclude that the multifunctional RuNPs may form the basis of new strategies on the treatment of liver cancer and other malignancies.

Introduction

2


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Liver cancer is one of the most prevalent malignant tumors worldwide. The most common type of liver cancer is Hepatocellular carcinoma (HCC), that is currently the third-leading cause of cancer death worldwide.1,2 Approximately 700,000 new cases of HCC occur annually worldwide, and most new cases occur in developing countries. The 5-year survival rate of HCC patients remains quite low, ranging between 6 and 11%, probably because of late diagnoses, resistance to treatment, tumor recurrence, and metastasis.3 Despite some progress in the treatment of liver cancer, existing therapies approved for clinical use by the US Food and Drug Administration, including cytotoxic drugs (estramustine, oxaliplatin, and doxorubicin), anti-angiogenic drugs such as sorafenib, bevacizumab, sunitinib, and thalidomide, and transarterial chemoembolization.4-10 However, there exist various serious adverse effects in the applications of these treatment strategies, and the drug resistance, relapse, and metastasis are still unresolved problems.10–11 For example, the current cytotoxic chemotherapy for liver cancer shows lower specificity and efficacy. Only 2–5% of therapeutic drugs are taken up by tumors, whereas more than 90% of the drugs accumulate in normal tissues.12,13 The treatment of anti-angiogenesis drugs sometimes may increase the risk of local invasion and metastasis, which contributes to the deterioration of cancer. 14 Therefore, the development of new therapeutic agents with effective control of the growth, proliferation and invasion of liver tumors has been receiving considerable attention.

Cancer nanotechnology is an emerging interdisciplinary field involving biology, chemistry, pharmaceutical engineering and medicine, has been applied widely. Nanosystems with unique style and integrated technology have considerable promise for the detection, prediction, prevention, and targeted therapy of cancer. Nanoparticles can be functionalized, 3



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and they may interact with biosystems in a more effective manner to regulate biological processes in disease development. Among them, one of the most notable is that targeting ligands can be attached to the nanoparticles, which is specificity for target cancer cells without causing substantial untoward effects on normal tissues.15-18 Nanosystems themselves with diagnostic or therapeutic properties can be designed to carry multiple therapeutic “payloads” through hydrogen bonds, van der Waals forces, hydrophobic–hydrophilic interactions, or static interactions. In addition, the nanosystems can bypass traditional drug resistance mechanisms. The nanodevices can provide essential breakthroughs in the fight against cancer.

A huge challenge in cancer nanotechnology is the mechanism for selectively delivering nanomaterials to tumor tissues while minimizing side effects in normal tissues. Targeting ligands play a crucial role in the targeted delivery of nanoparticles to tumor sites.19,20 Epigallocatechin gallate (EGCG) is the most abundant and active catechin in green tea, which is known as a particularly potent natural antioxidant beverage. EGCG has been demonstrated to suppress the tumor formation and progression in different organs, including the lungs, stomach, skin, liver, breast, pancreas, and prostate.21-23 EGCG has anti-matrix metalloproteinase, anti-proliferative, and apoptotic activities, in addition to the ability to inhibit cell invasion, angiogenesis, and metastasis.24,25 Recently, Nano-EGCG has been shown to impart antiproliferative and chemopreventive effects against several cancers.26-28 Encapsulated EGCG alone or in combination with cisplatin was more effective in inhibiting cell metastasis, angiogenesis.29 EGCG functionalized radioactive gold nanoparticles show efficacy in treating prostate cancer.30 Interestingly, EGCG has been demonstrated to directly 4


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bind to the 67-kDa laminin receptor (67LR) with an excellent specificity and selectivity.31 67LR as a cell surface EGCG receptor is a nonintegrin-type cell surface receptor with a high affinity for laminin. The receptor was first isolated from human breast carcinoma and murine melanoma cells.32 It is not only an important molecule in cell adhesion to the extracellular matrix, but also intimately associated with the invasion and metastasis of tumor cells. Importantly, 67LR is overexpressed in HCC cells.33,34

In a recent study, we found that ruthenium complexes manifest higher efficiency for inhibiting tumor growth and angiogenesis, particularly tumor metastasis,35-39 and they thus have potential applications in the field of cancer therapy. Notably we found that Se/Ru nanoparticles can suppress metal-induced Aβ40 aggregation in subjects with Alzheimer's disease with few side effects.40 In this study, we attempted to improve the targeting of 67LR by preparing novel ruthenium nanoparticles (EGCG-RuNPs) using EGCG as reducing and capping agents. Subsequently, luminescent ruthenium complexes were used to modify the surface of the nanoparticles via electrostatic adsorption. As expected, in SMMC-7721 cells overexpressing 67LR, our synthesized nanoparticles selectively bound with high affinity to 67LR and mainly accumulated in the cytoplasm of SMMC-7721 cells, and improved anti-cancer activity without causing substantial untoward effects on normal tissues. This would greatly enhance both the survival and quality of life of patients.

Materials and methods

Reagents and cells

All reagents and solvents were purchased commercially unless otherwise specified and 5



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doubly distilled water was used in this study. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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Ruthenium(III) chloride, EGCG, bromide

(MTT),

20,

70-dichlorofluorescein diacetate (DCFH-DA) and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich Chemical Co. Caspase-3 substrate (Ac-DEVD-AMC) was purchased from

Biomol (Germany),

Caspase-9

(Ac-LEHD-AFC)

and

caspase-8

substrates

(Ac-IETD-AFC) from Calbiochem. The human HCC line SMMC-7721 and liver cell line L-02 cells were purchased from the Chinese Academy of Sciences Cell Bank. All the solvents were of HPLC grade while the other chemicals used were of analytical grade. All cells were cultured in a highly humidified atmosphere with 5% CO2 at 37°C. Other chemicals were purchased from Sigma.

Synthesis of [Ru(bpy)2(4-B)] (ClO4)2·2H2O (RuBB)

Ruthenium (III) chloride hydrate was purchased from Alfa Aesar, and 1,10-phenanthroline-5,6-dione，2,2'-Bipyridine，and 4-formylphenylboronic acid were obtained from Sigma. The compound of cis-[Ru(Bpy)2Cl2].2H2O was synthesized by the reaction with Ruthenium (III) chloride hydrate, 2,2'-Bipyridine, and lithium chloride. The full synthesis details of 2-[4-(dihydroxyboryl)phenyl]-imidazo-[4,5-f][1,10]phenanthroline(4-B) and [Ru(Bpy)2(4-B)] (ClO4)2•2H2O (RuBB) were according to methods in the supporting information.

Synthesis of RuBB-loaded EGCG-RuNPs

The solution of 5 mM EGCG was freshly prepared in Milli-Q water. The solution of 5 mM 6


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RuCl3 was prepared by dissolving 0.1035 g RuCl3 powder in 100 mL Milli-Q water. The RuBB-loaded EGCG-RuNPs were synthesized as follows: First, 4 mL EGCG solution was added into 1 mL of 5 mM RuCl3 solution under magnetic stirring. The mixed solution was heated to 40℃ for the synthesis of RuNPs at pH 3. Then, 2 mg RuBB was added to mixed solution, the reaction mixture was sonicated (Branson Inc, USA) for 2 h. Then, The excess reactants were eliminated by centrifuge. The particles were washed three times with Milli-Q water followed by three times with anhydrous ethanol, with sonication and centrifugation (8000 rpm, 10 min) after each washing cycle. Finally no Ru was detected in the outer solution by ICP-AES. The synthesized RuBB-loaded EGCG-RuNPs were dissolved with Milli-Q water for experiments. The micrographs of RuBB-loaded EGCG-RuNPs were obtained on Hitachi (H-7650) for TEM. The elemental composition of nanoparticles was measured on an EX-250 system (Horiba) for TEM-EDX. The zeta potential and size distribution of nanoparticles were measured by PCS on a Nano-ZS instrument (MalvernInstruments Limited).

Live cell confocal microscopy

SMMC-7721 cells were grown on chamber slides to 70% confluence. RuBB-loaded EGCG-RuNPs (20 µg/mL) were added to the culture medium (final DMSO concentration, 0.1% v/v) and incubated for different time intervals at 37°C and nuclei were stained by Hoechst 33324 (10 µM) for 10 min. The cells were rinsed with PBS. Confocal images were captured with a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) using RuII (480 nm excitation, detection at 560-620 nm (green) and 625-754 nm

7



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(red)).

To investigate whether 67LR participated in the internalization of RuBB-loaded EGCG-RuNPs into SMMC-7721 cells, the cells were incubated with 5 µg/mL 67LR-blocking antibody (MLuC5) or 5 µg/mL laminin at 37°C in 5% CO2 for 1 h, and then with RuBB-loaded EGCG-RuNPs (10 µg/mL) for 2 h before imaging.

For further analysis of energy-dependent pathways, the cells were incubated with 10 µg/mL RuBB-loaded EGCG-RuNPs (10% PBS: 90% serum-free media) at 4°C, 25°C and 37°C for 1 h, respectively. To further explore the internalization mechanisms and uptake pathways of RuBB-loaded EGCG-RuNPs, cells were treated with endocytic inhibitors (50 µM chloroquine or 100 mM NH4Cl) at 37°C in 5% CO2 for 30 min, and then with RuBB-loaded EGCG-RuNPs (10 µg/mL) for 2 h before imaging.

Cellular uptake of RuBB-loaded EGCG-RuNPs

In order to quantitatively investigate the binding affinity of RuBB-loaded EGCG-RuNPs for 67 LR, SMMC-7721 cells in growth medium were seeded in 35 mm tissue culture dishes (Corning) and incubated at 37°C, under a 5% CO2 atmosphere until 70% confluent. The cells were incubated with 15 µg/mL 67LR-blocking antibody (MLuC5) or 15 µg/mL laminin for 1 h, followed by 4 h incubation with nanoparticles. The cell was trypsinized and rinsed twice with ice cold PBS. The samples were analyzed by a FACSCalibur flow cytometer (Becton Dickinson & Co., Franklin Lakes, NJ) with excitation at 480 nm.

Preparation of cells for transmission electron microscopy (TEM) 8


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SMMC-7721 cells (2×105 cells/mL ) were grown in DMEM medium containing 10% FBS at 37°C，and in a 5% CO2 atmosphere. Afte incubation with 20 µg/mL RuBB-loaded EGCG-RuNPs for 12 h, cells were thoroughly washed with PBS to eliminate RuBB-loaded EGCG-RuNPs that were not internalized. After cells were centrifuged, the culture medium was decanted. Cells were fixed in a 1 mL 2.5% gluteraldehyde solution for 2 h. Cells were then washed with 0.1M PBS for three times and postfixed in 1% osmium tetroxide solution for 1 h, washed with 0.1M PBS for three times, stained with 0.5% uranyl acetate for 1 h, dehydrated in a graded series of ethanol (50, 70 and 90 %) and acetone (90 and 100 %) TEM samples were embedded in epoxy resin and imaged under a 120 kV FEI Tecnai Spirit TEM.

MTT assay SMMC-7721 and L-02 cells were seeded on 96-well plates (5.0×103/well) and exposure to various concentrations of compounds. The microplate was incubated for 48 h at 37°C, 5% CO2.. Cells were incubated with 10 µL of MTT reagent (5 mg/mL) for 2 h，and then incubated with DMSO (150 µL for each well). Finally, the optical density (OD) at 570 nm was recorded. All drug doses were parallel tested in triplicate .

Cell migration and invasion assay Cell migration was determined using the wound-healing method. SMMC-7721 cells (6×105 cells/mL) were allowed to grow into a full confluence in 6-well plates precoated with 0.1% gelatin and then incubated with 10 µg/mL mitomycin C at 37°C, 5% CO2 for 2 h to inactivate SMMC-7721 cells. The confluent cell monolayers were wounded by scratching 9



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with 1 mL pipette tip. After being washed with PBS twice, cells were treated with various concentrations of complexes. Images were taken by Nikon digital camera after 24 h of incubation at 37°C, 5% CO2. At least three independent experiments were performed.

For cell invasion assay, their invasive ability was determined using matrigel coated transwell chambers (Corning, USA). The top chambers were seeded SMMC-7721 cells ( 1×105 cells/well) in 200 µL serum-free medium, and the lower chambers were filled with 500 µL medium containing 10% FBS. After incubation in the culture medium (control) or with RuBB-loaded EGCG-RuNPs at 5, 10 µg/mL, RuBB, RuNPs and EGCG at 20 µg/mL for 12 h, SMMC-7721 cells on the upper surface of the membrane (non-invasive cells) were wiped with cotton swabs and cells spreeding on the bottom side of the chamber (invasive cells) were fixed by paraformaldehyde, stained with H&E and counted in three random fields per chamber by microscope.

Measurement of intracellular reactive oxygen species (ROS) generation

RuBB-loaded EGCG-RuNPs induced ROS in SMMC-7721 and L-02 cells were monitored by DCF-DA assay. SMMC-7721 cells were seeded (1×106 cells/mL) into six-well plate and then incubated with 10 µM DCFH-DA at 37°C for 30 min. The cells were incubated with 20 µg/mL RuBB-loaded EGCG-RuNPs or 5 µg/mL doxorubicin at 37°C for 8 h. In another group of cells, pretreatment of N-acetylcysteine (NAC 5 mM) was done for 4 h followed by RuBB-loaded EGCG-RuNPs (20 µg/mL) for 8 h. DCF fluorescence was recorded on laser scanning confocal microscopy with excitation and emission wavelengths at 495 nm and 525 nm, respectively (Nikon Eclipse 80i). 10


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For glutathione S-transferase (GSH) measurement, SMMC-7721 cells were seeded (1×106 cells/mL) into six-well plate and then treated with RuBB-loaded EGCG-RuNPs (20 µg/mL) for 8 h in the presence or absence of NAC (5 mM). After treatment,, cells were washed with PBS and incubated with 30 µM GSH reactive dye CMFDA (Invitrogen) for 30 min at 37°C in the dark. CMF fluorescence was detected on fluorescence microplate reader with excitation and emission wavelengths at 485 and 535 nm, respectively.

Flow cytometric analysis

Induction of apoptosis by RuBB-loaded EGCG-RuNPs was quantified by flow cytometric analysis. Cells (4 × 105 cells/mL) were cultured in six-well plates and treated with 10 µg/mL RuBB-loaded EGCG-RuNPs for 24 h or NAC alone for 2 h. Another group of cells was prepared where pretreatment of NAC was done for 2 h followed by RuBB-loaded EGCG-RuNPs (10 µg/mL) for 24 h, collected, and washed twice with PBS. To detect early and late apoptosis, as described in our previous paper,38 after treatment, cells were analyzed using a FACS Calibur (BD Biosciences).

Determination of caspase activity

Caspase activities were measured using Caspase Activity Kit according to the manufacturer's instructions. Briefly, cells treated by nanoparticles were washed twice with ice-cold PBS, left on crushed ice for 15 min and then centrifuged at 4°C for 15 min. Subsequently, specific caspase substrates (Ac-DEVD-AMC for Caspase-3, Ac-IETD-AMC for caspase-8 and Ac-LEHD-AMC for caspase-9) were added and incubated at 37°C for 1 h. Caspase activity was determined using fluorescence intensity with the excitation and 11



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emission wavelengths set at 380 nm and 440 nm, respectively.

Xenograft mouse model

The 5–6-week-old severe combined immune deficiency (SCID) male mice (ordered from NIH) weighing ~ 20 g were divided into groups with five mice per group. The SMMC-7721 cells (1×107 cells per mouse) were implanted into the rear flank of nude mice (0.2 ml/mouse) to form a solid tumor. After the tumors had become established (~50 mm3), the mice were intratumorally injected with 2.5 mg /kg RuBB-loaded EGCG-RuNPs and PBS (control) every day. The tumor sizes were determined by Vernier caliper measurements and calculated as length×width×height. The body weights of male mice were recorded every day. After two weeks, the mice were sacrificed when the tumor volumes were noticeable. The weight of the mice and the tumor volume were recorded. Animal care was in accordance with the institutional guidelines. and approved by the Animal Care and Use Committee of Jinan University. Statistics

Statistical significance was estimated using one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. Statistical significance was set at p < 0.05

Results and discussion

Synthesis and characterization of RuBB-loaded EGCG-RuNPs

EGCG-functionalized RuNPs loaded with luminescent ruthenium complexes were successfully synthesized via a simple one-pot method in the presence of 0.1 mM RuBB 12


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(Figures S1-3, in Supporting Information) (Figure 1A). The redox potentials of RuCl52-/Ru [+0.68 V vs. Standard Hydrogen Electrode (SHE)] is more positive than that of EGCG (+0.42 V vs. SHE).30 Therefore, EGCG has the capacity to reduce Ruthenium (III) chloride to ruthenium atoms. The yield of RuBB-loaded EGCG-RuNPs was 6.1 mg (calculated by 10.35 mg RuCl3), and contained 35.23% RuBB calculated by fluminescence intensity of RuBB (Figures S3), In this reaction, ruthenium(III) salt was converted into Ru(0)NPs by EGCG (Figures S4), which served as both reducing and capping agents. The morphology of the as-prepared RuBB-loaded EGCG-RuNPs was firstly characterized by TEM. As shown in TEM images (Figure 1B), RuBB-loaded EGCG-RuNPs were monodisperse and homogeneous spherical nanoparticles with diameters of 30–90 nm and an average size of 73.59 nm (Figure 1C), which particles smaller than 100 nm help to avoid the RES system, and enter into disease organs easily. The zeta potential values of unloaded- and RuBB-loaded EGCG-RuNPs were −33.8 and −17.9 mv, respectively (Figure 1D), suggesting that positively charged RuBB2+ was loaded onto the surface of ECGC-RuNPs by electrostatic adsorption. Further investigation via surface elemental composition analysis employing EDX (Figures 1E–F) indicated that under the same conditions, the presence of a signal from Ru atoms increased from 19.87 to 28.38% after loading with RuBB. Other minor signals of C (32.84%) and O (13.86%) could be attributed to the elements of EGCG (Figure 1E), and the C (37.33%) and O atom signals (7.62%) could be attributed to the elements of EGCG and RuBB (Figure 1F). The Cu signal could be attributed to the copper mesh holding the TEM sample.

Stability of nanomaterials is important for its medicinal applications. Therefore, the size 13



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distribution of nanoparticles was measured after being put into PBS and cell culture mediumat at room temperature for 6 days. The results show that RuNPs alone were unstable in PBS and the cell culture medium (Figure 1G). Average particles size of RuNPs increased dramatically from 35.9 ±2.5 nm to 989 ±20.1 nm under physiological conditions (PBS, pH = 7.4), while from 40 ±2.3 nm to 1170 ±25.5 nm in the cell culture medium. By contrast, the RuBB-loaded EGCG-RuNPs remained stable for at least 6 days under these conditions, with the size ranging from 74 to 125 nm. These results suggest the high stability and application potential of RuBB-loaded EGCG-RuNPs in medicine.

67LR mediated the cellular internalization of RuBB-loaded EGCG-RuNPs

67LR is overexpressed in a variety of tumor cells. EGCG binds to 67LR in cancer cells in a concentration-dependent manner. Additionally, it has been demonstrated that 67LR is involved in the invasive and metastatic processes of HCC.33,34 Firstly, as shown in Figures 2, confocal images showed that the high photoluminescence signals of the RuBB-loaded EGCG-RuNPs were observed in cells, thus the cellular uptake properties of RuBB-loaded EGCG-RuNPs can be studied by flow cytometry and confocal microscopy conveniently.41,42 To investigate the binding affinity of RuBB-loaded EGCG-RuNPs for 67LR, which is overexpressed in SMMC-7721 HCC cells, in vitro 67LR binding assays were performed. First, to investigate whether 67LR participated in the internalization of RuBB-loaded EGCG-RuNPs into SMMC-7721 cells, receptor blocking studies were performed by confocal luminescence microscopy. Initially, SMMC-7721 cells were preincubated with laminin or 67LR-blocking antibody (MLuC5) at room temperature for 1 h. Laminin, a 14


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known ligand for 67LR. MLuC5, which specifically recognizes the 67-kD high affinity laminin receptor molecule. As shown in Figures 2A–B, intracellular luminescence was significantly suppressed by treatment with 67LR-blocking antibody or laminin before the addition of RuBB-loaded EGCG-RuNPs, whereas these receptor inhibitors had no effect on the uptake of RuBB. These results suggested that the uptake of RuBB-loaded EGCG-RuNPs into SMMC-7721 cells was mediated by 67LR, whereas 67LR didn’t participate in the internalization of RuBB into SMMC-7721 cells, which implied the RuBB binding to the nanoparticles are stable. In addition, luminescence of 20 µg/mL Ru-BB in the cells was close to that of 10 µg/mL RuBB-loaded EGCG-RuNPs (containing 35.23% RuBB), which implied the absorption efficiency of RuBB-loaded EGCG-RuNPs was significantly higher than that of RuBB in SMMC-7721cells.

To further explore the cellular entry pathway of RuBB-loaded EGCG-RuNPs, we investigated

whether

the

uptake

of

RuBB-loaded

EGCG-RuNPs

was

due

to

energy-dependent pathways. In this respect, RuBB-loaded EGCG-RuNPs at a concentration of 10 µg/mL were incubated with SMMC-7721 cells at 4, 25, and 37°C, and the relative uptake of the nanoparticles was assayed by confocal luminescence microscopy. As shown in Figures 2C–D, the cellular uptake of RuBB-loaded EGCG-RuNPs was significantly reduced at 4 and 25°C, and the cellular uptake of RuBB-loaded EGCG-RuNPs increased with temperature, indicating that the process was energy-dependent. Endocytosis serve many important cellular functions including the uptake of nanomaterials, and it is an energy-dependent uptake route.43 Thus, the endocytic inhibitors chloroquine and NH4Cl were used to examine the internalization mechanisms and uptake pathways of RuBB-loaded 15



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EGCG-RuNPs in SMMC-7721 cells. As shown in Figure 2C, chloroquine and NH4Cl strongly decreased the luminescence intensity of RuBB-loaded EGCG-RuNPs in SMMC-7721 cells to 55.2 and 46%, respectively, of the control level (Figure 2D), suggesting that the main pathway for their internalization was endocytosis.

To further quantitatively estimate the binding affinity of RuBB-loaded EGCG-RuNPs for 67LR, we measured the internalization of the nanoparticles by flow cytometry. As shown in Figures 3A and 3C, after SMMC-7721 cells without pretreatment were incubated with 10 µg/mL RuBB-loaded EGCG-RuNPs for 0, 1, 2, and 4 h, the luminescence intensity of cells was significantly increased in a time-dependent manner, reflecting the highly efficient internalization of RuBB-loaded EGCG-RuNPs into SMMC-7721 cells. There was a marked decrease in the internalization of RuBB-loaded EGCG-RuNPs into SMMC-7721 cells that were pretreated with laminin or 67LR-blocking antibody as compared with that in unblocked cells (Figure 3B). Laminin inhibited the cellular uptake of RuBB-loaded EGCG-RuNPs at 4 h by 50% relative to the unblocked level, whereas 67LR-blocking antibody inhibited uptake by as much as 80% (Figure 3C). Furthermore, we compared the cellular uptake of RuBB-loaded EGCG-RuNPs and RuBB at the same concentration and incubation time. RuBB-loaded EGCG-RuNPs had stronger luminescence intensity than RuBB at 4 h (Figure 3D). In addition, the cellular uptake of RuBB was not affected by these receptor inhibitors (Figure 3E-F). These results were consistent with the confocal microscopy results, suggesting that the highly efficient cellular uptake of RuBB-loaded EGCG-RuNPs into SMMC-7721 cells was mediated by 67LR and verify our hypothesis that RuBB-loaded EGCG-RuNPs were targeted to cancer cells via surface functionalization with 16


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EGCG.

Localization of RuBB-loaded EGCG-RuNPs in vitro

TEM images revealed that abundant amounts of the 20 µg/mL nanoparticles were internalized into SMMC-7721 cells and were observed in the cytoplasm and vacuoles without entering the nucleus (Figure 4A). In addition, as shown in Figure 4B, RuBB-loaded EGCG-RuNPs could interact with proteins located in the cytoplasm, and they were found to be confined to endocytic vesicles. This suggests that the predominant route of cellular uptake of the nanoparticles is endocytosis.

To investigate the intracellular behavior of RuBB-loaded EGCG-RuNPs, confocal microscopy was used to monitor the time-dependent accumulation of RuBB-loaded EGCG-RuNPs conjugations in SMMC-7721 cells (Figure 5). Cells incubated with 20 µg/mL RuBB-loaded EGCG-RuNPs showed two types of fluorescence. As shown in Figure 5, RuBB-loaded EGCG-RuNPs were found accumulate in the cytoplasm of SMMC-7721 cells after 6 h, at which point green/red fluorescence dots were not observed in the nucleus. With the incubation time prolonged, the turquoise colocalization areas of the green fluorescence from RuBB-loaded EGCG-RuNPs and the blue fluorescence from nucleus were observed, while purple colocalization areas of the red fluorescence from RuBB-loaded EGCG-RuNPs and the blue fluorescence from nucleus were also observed (Figure 5). The overlapped areas increased with the incubation time from 6 to 12 h, which implied a gradual accumulation of RuBB-loaded EGCG-RuNPs into the nucleus. The nucleus may be a potential target organelle of RuBB released from NPs. Taken together, the aforementioned 17



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data indicate that the 67LR-mediated endocytotic pathway is involved in the cellular uptake of RuBB-loaded EGCG-RuNPs into SMMC-7721 cells, but it is not responsible for the uptake of RuBB, which implied RuBB-loaded EGCG-RuNPs are internalized by selective uptake in liver tumors. Energy plays an extremely important role in the process by which RuBB-loaded EGCG-RuNPs cross the plasma membrane and eventually reach the nucleus. These findings sparked us to do further detailed antitumor efficacy studies of RuBB-loaded EGCG-RuNPs.

Cytotoxicity of RuBB-loaded EGCG-RuNPs

To evaluate the tumor cytotoxicity of RuBB-loaded EGCG-RuNPs, the concentrations of 0, 2.5, 5, 10 and 20 µg/mL for the nanoparticles were incubated with SMMC-7721 and L-02 cells for 48 h, respectively, and cell viability was determined by the MTT assay. As shown in Figure 6A, RuBB-loaded EGCG-RuNPs significantly suppressed the growth of SMMC-7721 cells in a concentration-dependent manner（IC50=4.41±0.63 µg/mL）, whereas the nanoparticles were noncytotoxic toward normal L-02 cells at concentrations as high as 100 µg/mL (Figure 6B and S5), suggesting that the RuBB-loaded EGCG-RuNPs were targeted to liver cancer cells, and show no significant cytotoxicity to normal cells.

To examine the anticancer effect of EGCG-functionalized ruthenium nanoparticles loaded with luminescent ruthenium complexes in SMMC-7721 cells, we further compared the cytotoxicity of RuBB-loaded EGCG-RuNPs, RuBB, RuNPs, and EGCG. As shown in Figure 6A, all four agents inhibited the proliferationof SMMC-7721 cells in a concentration-dependent manner, but RuBB-loaded EGCG-RuNPs and RuNPs showed 18


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greater inhibitory effects than those of RuBB and EGCG. In contrast to RuNPs alone (10 µg/mL; 45.9%), RuBB-loaded EGCG-RuNPs at a concentration of 10 µg/mL significantly decreased cancer cell viability to 21.2% of the control level. This indicates that EGCG surface decoration is the key factor to enhance the anticancer effect of RuBB-loaded EGCG-RuNPs in SMMC-7721 cells.

Effect of RuBB-loaded EGCG-RuNPs on the migration and invasion of SMMC-7721 cells

Cell invasion and migration are characteristic features of HCC and the main cause of death in patients with HCC.44 Therefore, we examined the effect of RuBB-loaded EGCG-RuNPs on the invasion and migration of SMMC-7721 cells. Cell migration was evaluated by the wound scratch assay, the results of which (Figure 7A) showed that the wounds in the control group were almost fully repopulated with migrated cells, whereas a significant area of the wound remained uncovered in RuBB-loaded EGCG-RuNPs-, RuNPs-, or RuBB-treated cells. Conversely, a small area remained uncovered in EGCG-treated cells. RuBB-loaded EGCG-RuNPs at a concentration of 5 and 10 µg/mL significantly inhibited cell migration by 58.6% and 81% relative to the control group, respectively (Figure 7B). The results from the Transwell invasion assay (Figure 7C) revealed that treatment with RuBB-loaded EGCG-RuNPs (5, 10 µg/mL), RuNPs (20 µg/mL), RuBB (20 µg/mL), or EGCG (20 µg/mL) inhibited the penetration of the Matrigel-coated filter by SMMC-7721 cells. After treatment with 5 and 10 µg/mL RuBB-loaded EGCG-RuNPs for 12 h, the invasive capacity of SMMC-7721 cells was significantly decreased by 70.8% and 85% relative to the control level, respectively. These results indicated that compared with RuNPs, RuBB, and EGCG,

19



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RuBB-loaded EGCG-RuNPs most strongly inhibited liver cancer cell migration and invasion.

Effect of RuBB-loaded EGCG-RuNPs on reactive oxygen species (ROS) generation and glutathione S-transferase (GSH) levels

ROS participate in the initiation and progression of a variety of human diseases. To study the potential role of ROS in-duced by RuBB-loaded EGCG-RuNP on the migration and invasion of tumor cells, the ROS generation in SMMC-7721 and L-02 cells was measured using the ROS-sensitive probe DCFH-DA. Based on our previous studies,38,39 fluminescence intensity of ROS-sensitive probe DCFH-DA was significantly stronger than that of RuBB-loaded EGCG-RuNPs. In addition, fluorescence from ROS was monitored on laser scanning confocal microscopy with excitation and emission wavelengths at 495 and 525 nm, and RuII (480 nm excitation, detection at 560~620 nm). Thus, fluorescence from RuBB-loaded EGCG-RuNPs had no too much effect on the ROS measurements (Figures S6). The intracellular ROS level of L-02 cells was compared with that of SMMC-7721 cells, as shown in Figures 8A–B. The treatment of SMMC-7721 cells with RuBB-loaded EGCG-RuNPs caused a marked increase in ROS production in a concentration-dependent manner(Figure 8C). Specifically, ROS levels were elevated by 1.8-fold compared with the control level following treatment with 20 µg/mL RuBB-loaded EGCG-RuNPs, whereas no increase was detected in L-02 cells. It is particularly interesting that the intracellular ROS levels in both SMMC-7721 and L-02 cells were significantly increased after treatment with the chemotherapeutic ROS-producing drug doxorubicin as a positive control. 45,46 (Figures

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8A–B). These results revealed a pro-oxidant effect of RuBB-loaded EGCG-RuNPs in tumor cells, whereas normal cells appeared unaffected.

N-acetylcysteine (NAC) is a precursor of GSH and direct antioxidant. To further understand whether GSH was involved in the RuBB-loaded EGCG-RuNP-initiated ROS production in SMMC-7721 cells, changes in GSH levels were evaluated. As shown in Figure 8E, RuBB-loaded EGCG-RuNPs decreased intracellular GSH levels; GSH levels were decreased to 70% of the control level after treatment with RuBB-loaded EGCG-RuNPs at a concentration of approximately 20 µg/mL for 8 h. In the present study, the increased production of ROS and reduced GSH levels suggested that RuBB-loaded EGCG-RuNPs caused imbalances between the formation and clearance of ROS and induced oxidative stress, changes that may lead to the migration and invasion of tumor cells. We used the potent antioxidant NAC to further examine the involvement of ROS in these processes. NAC pretreatment suppressed the RuBB-loaded EGCG-RuNP-induced increase in ROS levels (Figure 8D) and decrease in GSH levels in SMMC-7721 cells (Figure 8E). It was interesting to note that NAC alone slightly reduced ROS levels but significantly increased GSH levels. There have been reported that the types of antioxidant enzymes and factors play a critical role in ROS and GSH production, for example, catalase, superoxide dismutase, NF-κB and HGF/SF, all of them can significantly affect the ROS and GSH production in cells at different levels46-49 Taken together, these results showed that RtuBB-loaded EGCG-RuNPs-induced oxidative stress probably plays a key role in migration and invasion in human SMMC-7721 cells.

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Induction of apoptotic cell death by RuBB-loaded EGCG-RuNPs

Intracellular ROS have been extensively demonstrated to play a crucial role in apoptosis induced by various anticancer agents. Further, to determine whether RuBB-loaded EGCG-RuNP-induced apoptosis in SMMC-7721 cells is induced by ROS, we pretreated cells with NAC, which is a ROS scavenger, before RuBB-loaded EGCG-RuNPs exposure and assessed the percentage of apoptotic cells. The results showed that NAC pretreatment significantly suppressed RuBB-loaded EGCG-RuNP-induced apoptosis in SMMC-7721 cells (Figure 9A). The percentage of apoptotic cells was significantly reduced from 29.3 to 14.1%, and the overall cell death rate decreased from 31.3 to 14.5% on NAC pretreatment (Figure 9A). This clearly suggested that cell apoptosis was induced as a result of ROS generation in response to RuBB-loaded EGCG-RuNPs exposure.

The process of apoptosis involves a series of cysteine proteases known as caspases, which are crucial in the initiation and execution of apoptosis.50 Among caspases, caspase-3 is considered as the most common executor of apoptosis, whereas caspase-8 is involved in initiating apoptosis through death receptor-mediated signaling pathways (extrinsic apoptosis pathway), and caspase-9 through mitochondria-mediated signaling pathways (intrinsic apoptosis pathway). To determine the possible signaling pathways involved in RuBB-loaded EGCG-RuNPs-induced cell death, the activities of caspase-3, caspase-8, and caspase-9 were detected by fluorometric assays. Figure 9C shows that the treatment of cells with 5 and 10 µg/mL RuBB-loaded EGCG-RuNPs significantly increased the activities of caspase-3, caspase-8, and caspase-9 in a concentration-dependent manner, suggesting that both the

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intrinsic

and

extrinsic

apoptotic

pathways

were

involved

in

RuBB-loaded

EGCG-RuNP-induced apoptosis. In detail, the activity of caspase-8 was elevated by 1.8-fold versus the control after exposure to 10 µg/mL RuBB-loaded EGCG-RuNPs for 24 h, whereas caspase-9 activity was increased by 2.4-fold, suggesting that compared with the extrinsic pathway, the intrinsic pathway contributed more to the induction of cell apoptosis.

Caveolin-1 downregulation by RuBB-loaded EGCG-RuNPs The role of ROS in cell invasion and migration has been described.51,52 Caveolin-1 (Cav-1) has received the most attention, as it has been demonstrated to be involved in cancer cell motility and tumor progression.53 Cav-1 plays an important role as a positive regulator of cancer cell migration and invasion. Cav-1 is also subject to regulation by ROS.54,55 O2 and H2O2 suppress the expression of Cav-1, and OH has an inhibitory effect on Cav-1 expression. To further determine the effect of RuBB-loaded EGCG-RuNP-initiated ROS on the invasion and migration of tumor cells, cells were treated with RuBB-loaded EGCG-RuNPs, and Cav-1 expression was detected by Western blotting. Figure 10 shows that incubation of cells with exogenous H2O2 for 24 h almost completely abrogated Cav-1 expression. The treatment of cells with 10 µg/mL RuBB-loaded EGCG-RuNPs significantly downregulated the expression of Cav-1 compared with the control level, whereas pretreatment with the antioxidant NAC upregulated the expression of the protein compared with that in RuBB-loaded EGCG-RuNP-treated cells. These data indicated the involvement of RuBB-loaded EGCG-RuNP-initiated ROS production in lowering the invasion and migration capacity of SMMC-7721 cells.

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In vivo antitumor efficacy

In an attempt to evaluate the anti-tumor efficacy of RuBB-loaded EGCG-RuNPs in vivo, a liver tumor (SMMC-7721) xenograft model was used. The untreated control group was injected with the same amount of PBS. As shown in Figure 11A, treatments started when the average tumor volume reached ~50 mm3. The control group exhibited a rapid increase in the tumor volume as a function of time, the average tumor volume increased to 1082.32 ±78.50 mm3 after 15 days. Notably, minimal increase in the tumor volume was found for the group treated with 2.5 mg kg-1 d-1 RuBB-loaded EGCG-RuNPs, the average tumor volume only increased to 255.01 ±39.64 mm3 after 15 days (Figure 11C), a significant inhibition in tumor growth. In addition, Figure 11B shows the variation in the average body weight of the mice over time. The body weight of the RuBB-loaded EGCG-RuNPs -treated animals was not different from that of the control groups (Figure 11B), suggesting that no apparent critical cytotoxicity was caused by the treatment with RuBB-loaded EGCG-RuNPs. Further, tumor weight in RuBB-loaded EGCG-RuNPs group (0.098 ± 0.03 g) is significant lower that in control group (0.391 ± 0.06 g) (Figure 11C-D), suggesting the high anti-tumor efficacy. Conclusion

Using EGCG as reducing and capping agents, Ru(II)-stabilized RuNPs with enhanced antitumor activity in SMMC-7721 cells were successfully synthesized through a simple and novel synthetic route. Because EGCG has a receptor binding ability, it can be used for the targeted delivery of nanoparticles to cells overexpressing 67LR. First, we successfully synthesized EGCG-RuNPs using a simple redox system. Furthermore, luminescent

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ruthenium complexes with anti-cancer efficacy were designed and bound to the surface of EGCG-RuNPs via electrostatic adsorption. RuBB-loaded EGCG-RuNPs showed small particle size with narrow distribution, better stability, and high selectivity between liver cancer and normal cells. We evaluated the specific targeting features of RuBB-loaded EGCG-RuNPs using HCC cells through in vitro 67LR binding assays. The results indicated that 67LR-mediated endocytosis played an important role in the uptake of RuBB-loaded EGCG-RuNPs into HCC cells. In addition, TEM and confocal microscopic images showed that RuBB-loaded EGCG-RuNPs accumulated in the cytoplasm of SMMC-7721 cells, the nucleus may be a potential target organelle of RuBB released from NPs. Furthermore, we demonstrate the anti-tumor efficacy of RuBB-loaded EGCG-RuNPs both in vitro and in vivo to solid tumors after systemic administration in a mouse tumor-xenograft model. Concentrations of RuBB-loaded EGCG-RuNPs that were nontoxic in normal L-02 cells showed direct ROS-dependent cytotoxic, proapoptotic, and anti-invasive capacity in SMMC-7721 cells. Moreover, ROS-dependent apoptosis in SMMC-7721 cells occurred through the activation of both the intrinsic and extrinsic apoptotic pathways. Importantly, RuBB-loaded EGCG-RuNPs exhibited greater efficacy than EGCG, RuNPs, and Ru(II)-polypyridyl complexes in treating liver cancer.

In comparison to other EGCG nanoparticles, RuBB-loaded EGCG-RuNPs exhibited comparatively high antitumor effects and also provides a stable fluorescent signal for intracellular imaging. For instance, EGCG encapsulated in PLA-PEG nanoparticles was evaluated against the 22R ν1 tumor growth using a xenograft model. They observed that nano-EGCG (100 µg/mice) inhibited 50% as compared with tumor control after 45 days.26 25



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EGCG-pNG (2 mg/mouse) inhibit 25% MBT-2 tumor growth in a C3H/He mouse model after 15 days.56 Whereas RuBB-loaded EGCG-RuNPs (50 µg/mice) decreased the average tumor volume to 23% of the control level after 15 days. Although further research, including clinical studies, is needed to confirm our findings, these data could serve as the basis of the development of new treatment modalities for liver cancer.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (21171070, 21371075), the Natural Science Foundation of Guangdong Province (2014A030311025, S2 013010011660).and Chinese Postdoctoral Science Foundation.

Supporting Information Available: Description of the material. This material is available free of charge via the Internet at

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Properties

of

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Novel

Ruthenium(II)

Complex,

J.

Inorg.

Biochem.

2004,98(6):1017-1022. (60) Zhang, Q. L.; Liu, J. H.; Ren, X. Z.; Xu, H.; Huang, Y.; Liu J. Z.; Ji L. N. A Functionalized Cobalt(III) Mixed-Polypyridyl Complex as a Newly Designed DNA Molecular Light Switch, J. Inorg. Biochem. 2003, 95, 194–198.

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Page 35 of 43

Figure

A

- -⊕ -⊕ -⊕ Ru - EGCG - ⊕ - - ⊕ -

RuCl3 +

C

D Total Counts

150000

15

100000

10 5

50000 0

100

-100

1000

0

O

7.62

Cu

33.43

Cu

26.67

Ru

19.87

Ru

28.38

Totals

100

Totals

100

O Ru

Ru

Cu

Cu

O

Ps

Ru

Cu

1000 200 100

1d 3d 6d

EG CG -

Ru

1200

-R uN Ps

13.86

EG CG

O

G

ad ed

37.33

BB -lo

Weight (%)

C

C

Ru N Ps

Element

32.84

Ru N

Cu

Weight (%)

C

Ru BB -lo ad ed

C

F

Element

Z-average (nm)

E

100

Zeta Potential (mV)

Size (nm)

Ru

200 nm 10 SSS

s

200nm

RuBB-loaded EGCG-RuNPs

200000

20

0

EGCG-RuNP

250000

25

Ru NP

B

Number (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 0

2

4

6

8

10

12

14

16

18

20 Kev

0

2

4

6

8

10

12

14

16

18

20 Kev

PBS

Cell culture medium

Figure 1 Preparation and characterization of RuBB-loaded EGCG-RuNPs. (A) Schematic illustration of the formation of RuBB-loaded EGCG-RuNPs. (B) TEM images of RuBB-loaded EGCG-RuNPs scale bar: 200 nm. (C) Size distribution of RuBB-loaded EGCG-RuNPs. (D) Zeta potential distribution of EGCG-RuNP and RuBB-loaded EGCG-RuNPs. EDX analysis of EGCG-RuNP (E) and RuBB-loaded EGCG-RuNPs (F). (G) Stability of RuNPs and RuBB-loaded EGCG-RuNPs in PBS (pH = 7.4) and cell culture medium

35



A

C

RuBB-loaded EGCG-RuNPs 10 µg/mL Unblocked

+ Laminin

RuBB-loaded EGCG-RuNPs 10 µg/mL

37℃

+Anti-67LR antibody

25℃

4℃

RuBB 20 µg/mL Unblocked

B

+ Laminin

+Colchicine 50 µM +NH +NH44Cl Cl100 100mM mM

+Anti-67LR antibody

D

120

120 Relative fluorescence Intensity( %)

100

Relative fluorescence Intensity( %)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 43

80 60 *

40

*

20 0

RuBB-loaded EGCG-RuNPs RuBB laminin Anti-67LR antibody

100 80

+ +

+ +

+

+ +

*

*

40 *

20 0

+

*

60

37

+ +

25

4

Chloroquine NH4Cl

(˚C)

Figure 2 Mechanism of cellular uptake of RuBB-loaded EGCG-RuNPs. (A) Receptor blocking studies of uptake of RuBB-loaded EGCG-RuNPs by SMMC-7721cells. The cells were pretreated with laminin or 67LR antibody（5 µg/mL）in the presence of RuBB-loaded EGCG-RuNPs（10 µg/mL）or RuBB（20 µg/mL）. (B) Relative luminescence intensity of RuBB-loaded EGCG-RuNPs and RuBB in SMMC-7721cells after laminin or 67LR antibody treatment. (C) Temperature-dependence studies of uptake of RuBB-loaded EGCG-RuNPs by SMMC-7721cells at 37, 25, 4°C (10 µg/mL) and treatment with the endocytosis inhibitors chloroquine (50 µM), NH4Cl (100 mM) displays in confocal luminescence image. (D) Relative luminescence intensity of RuBB-loaded EGCG-RuNPs in SMMC-7721 cells after Temperature-dependence and inhibition treatment. (*) p < 0.05 vs. RuBB-loaded EGCG-RuNPs (B), or vs. 37°C (D).

36


B


100

50

C Unblocked +Laminin +Anti-67LR

75

Counts

Counts

75

50

Counts

25

25 100

101

102

103

10 0

104

Emission intensity (A.U)

D

C


100

C 1h 2h 4h

10 2

10 3

E

75

100

RuBB

Counts

50 25

50 25

10 1

10 2

103


60

*

40

*

0

*

*

20

10 4

C Unblocked +Laminin +Anti-67LR

75

100

1h 2h 4h

80

*

*

Unblocked + Laminin +Anti-67LR


C RuBB RuBB-loaded EGCG-RuNP

100

10 1


100


A

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10 4

100

101

102

103

F

RuBB 120


Page 37 of 43

104

100 80 60 40 20 0

Unblocked + Laminin +Anti-67LR


Figure 3 Cellular uptake results of SMMC-7721 cells. (A)Time-dependent cellular uptake profiles of 10 µg /mL RuBB-loaded EGCG-RuNPs. (B) Cellular uptake of RuBB-loaded EGCG-RuNPs (10 µg/mL, 4 h) after laminin or 67LR antibody（15 µg/mL）treatment. (C) Relative luminescence intensity of RuBB-loaded EGCG-RuNPs in SMMC-7721 cells. (D) Cellular uptake of RuBB-loaded EGCG-RuNPs and RuBB (10 µg/mL, 4 h) (E) Cellular uptake of RuBB after laminin or 67LR antibody treatment. (F) Relative luminescence intensity of RuBB in SMMC-7721 cells after laminin or 67LR antibody treatment. (*) p < 0.05 vs. unblocked.

A

B

1µm

2µm

Figure 4 TEM image showing internalization of nanoparticles in SMMC-7721 cells. These cells incubated with （20 µg/mL） RuBB-loaded EGCG-RuNPs for 12 h. Their cellular locations are indicated by black arrows. Parts B is picture with higher magnification showing detailed structures.

37



Figure 5 Confocal fluorescence images showing localization of nanoparticles in SMMC-7721 cells . Confocal fluorescence images of RuBB-loaded EGCG-RuNPs (20 µg/mL) in SMMC-7721 cells stained with Hoechst 33342 (10 µM). ( red emission from ruthenium complex, excited at 480 nm and emitted at 625–754 nm; green emission also from ruthenium complex, excited at 480 nm and emitted at 560–620 nm; blue emission from Hoechst 33342 excited at 405 nm and emitted at 420-480nm.). scale bar: 10 µm.

140 120 100 80 60 40 20 0

#

*# *# *

B

RuBB-loaded EGCG-RuNPs RuBB RuNP EGCG

Cell viability ( % )

A Cell viability ( % )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 43

#

*#

*# *#

*# *#

*

*# *# *#

* *

0

2.5

5

10

20

Concentration ( µg/mL)

140 120 100 80 60 40 20 0

SMMC-7721 cells L-02 cells

*

* *

* *

* *

0

*

*

*

*

20

40

60

100

Concentration ( µg/mL)

Figure 6 Growth inhibition of RuBB-loaded EGCG-RuNPs in SMMC-7721 cells (A) and L-02 cells (B). Cells were treated with different concentrations of RuBB-loaded EGCG-RuNPs, RuNPs, RuBB and EGCG for 48 h. Cell viability was determined by the MTT assay as described in Materials and Methods. (*) p < 0.05 vs. control, or SMMC-7721 cells vs. L-02 cells (brackets); (#) p < 0.05 vs. RuBB-loaded EGCG-RuNPs .

38



24 h

Ru BB

60

*

*#

*#

*#

*

*

20 Control 5

10

20

RuBB

EGCG

20 µg/mL

20 µg/mL

20

20

120 100 80 60

*

40

*#

*#

*#

*

20 0

EG CG

CG

Ru BB

N Ps

80

Ru

-lo ad ed

EG

100

RuNPs 20 µg/mL

D

CG -R uN Ps

120

0

10 µg/mL

Ru BB

5 µg/mL

40

24 h

RuBB-loaded EGCG-RuNP

B

EGCG 20 µg/mL

CG -R uN Ps

Control

RuBB 20 µg/mL

Invasive cells (% of control)

C

24 h

RuNPs 20 µg/mL

s

24 h

10 µg/mL

EG

5 µg/mL

Ru NP

Control

EG

A

Migratory cells (% of control)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ru BB -lo ad ed

Page 39 of 43

* Control 5

µg/mL

10

20

20

20

µg/mL

Figure 7 Effects of RuBB-loaded EGCG-RuNPs, RuBB, RuNPs and EGCG on migration and invasion of SMMC-7721 cells. Typical images of the wound 24 h after incubation in the culture medium (control) or with RuBB-loaded EGCG-RuNPs at 5, 10 µg/mL, RuBB, RuNPs and EGCG at 20 µg/mL. Images of the invasion after incubation in the culture medium (control) or with RuBB-loaded EGCG-RuNPs at 5, 10 µg/mL, RuBB, RuNPs and EGCG at 20 µg/mL for 12 h (A) Images of wound healing assay displaying cell migration to the wound. (C) Images of Transwell invasion assay (with Matrigel) (magnification, 200×). Quantitative analysis of the inhibitory effects on migration (B) and invasion (D). After incubation, the cells were quantified by manual counting. These experiments were performed thrice with similar results and significant differences from control group were observed (* p < 0.05 vs. control or 5 µg/mL vs. 10 µg/mL RuBB-loaded EGCG-RuNPs (brackets); (#) p < 0.05 vs. 5 µg/mL RuBB-loaded EGCG-RuNPs). Data are presented as the percentages of the control group, which was set at 100%.

39



L-02

A

B

Control RuBB-loaded EGCG-RuNPs Doxorubicin

Control RuBB-loaded EGCG-RuNPs Doxorubicin

ROS Fluorescence Intensity ( % of control)

300

SMMC-7721 Control RuBB-loaded EGCG-RuNPs Doxorubicin

250

*

150 100 50

D

120


*

150

*

*

90 60 30 0

Control 2.5

5

10

20

RuBB-loaded EGCG-RuNPs ( µg/mL)

SMMC-7721

E *

*

180

L-02

180

180

GSH Fluorescence Intensity ( % of control)

C

*

*

200

0


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 43

150

*

120 90 60 30 0

RuBB-loaded EGCG-RuNPs NAC

*

150 120 90

*

60 30 0

+

+

+ +

RuBB-loaded EGCG-RuNPs NAC

+

+

+ +

Figure 8 Effect of RuBB-loaded EGCG-RuNPs on ROS and GSH levels in SMMC-7721 cells and its inhibition by NAC pretreatment. (A) SMMC-7721 and L-02 Cells incubated with 10 µM DCFH–DA in PBS for 30 min were treated with 20 µg/mL nanoparticles or 5 µg/mL doxorubicin for 8 h at 37°C, and then imaged under a fluorescence microscope. （B） Quantitative analysis of ROS levels in SMMC-7721 and L-02 Cells (C) Quantitative analysis of DCF fluorescence intensity of SMMC-7721 cells treated with different concentrations of nanoparticles. (D) Effects of NAC on ROS generation induced by nanoparticles. (E) Effects of NAC on GSH Levels induced by nanoparticles. Cells were pretreated with 5mM NAC for 4 h and then exposed to 20 µg/mL RuBB-loaded EGCG-RuNPs for 8 h. All results were obtained from three independent experiments. (*) p < 0.05 vs. control.

40


Page 41 of 43

Control

NAC


RuBB-loaded EGCG-RuNPs +NAC

0.1%

0.4 %

0.2%

0.9%

2.0%

22.2%

0.4%

8.1%

99.1 %

0.4 %

98.1 %

0.8 %

68.7%

7.1%

85.5%

6.0%

B

C

Live cells Apoptotic cells

120

*

100 80

*

* *

30 20

*

10 0 RuBB-loaded EGCG-RuNPs NAC

+

**

200

*

* *

150

**

*

100 50 0

+

Control 5 µg/mL 10 µg/mL

300 250

* Caspase Viability (%) of control

A

Cells (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


+ +

Caspase-3

Caspase-8

Caspase-9

Figure 9 RuBB-loaded EGCG-RuNPs treatment caused apoptosis in SMMC-7721 cells (A) Effects of NAC on apoptosis induced by 10 µg/mL nanoparticles. (B) Quantitative analysis of RuBB-loaded EGCG-RuNPs -induced apoptosis in the presence of ROS inhibitor NAC. (C) Cells were treated with 10 µg/mL RuBB-loaded EGCG-RuNPs for 24 h. Caspase activities were measured by a fluorometric method. All results were obtained from three independent experiments. (*) p < 0.05, (**) p < 0.01 vs. control, or RuBB-loaded EGCG-RuNPs vs. RuBB-loaded EGCG-RuNPs + NAC (brackets). Cav-1

β-actin

NAC RuBB-loaded EGCG-RuNPs H2O2

-

+

+ -

+ + -

Figure 10. Expression of caveolin-1 (Cav-1) in SMMC-7721 cells. Cells were treated with H2O2 (100 µM), RuBB-loaded EGCG-RuNPs (10 µg/mL) for 24 h , or pretreated with 5mM NAC for 4 h and then RuBB-loaded EGCG-RuNPs treated for 24 h. Cell lysates were prepared and analyzed for Cav-1 expression. H2O2 was used as a positive control and β41



A

1200

Control RuBB-loaded EGCG-RuNPs

1000 800 600 400 200 0

B

24

Body weight (g)

actin as a loading control. Three independent experiments were performed.

Tumor volume ( mm3)

20


16

12 0

3

6

9

12

15 (day)

0

C

D Tumor weight (g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 43


2

4

6

8 10 12 14 16 (day)

0.5 0.4 0.3 0.2

** 0.1 Control


Figure 11. RuBB-loaded EGCG-RuNPs inhibits tumor growth in vivo. SMMC-7721 cells (1×107 cells per mouse) were implanted into the 5- or 6-week-old SCID male mice. After the tumors had become established (~50 mm3), the mice were intratumorally injected with 2.5 mg /kg RuBB-loaded EGCG-RuNPs and PBS (control). After 2 weeks, mice were sacrificed and tumors were removed and taken images by Nikon camera. (A) Tumor volume, (B) average mouse body weight, (C) photograph of tumors and (D) tumor weight of control and nanoparticles treated group. (**) p < 0.01 vs. control.

42


Page 43 of 43

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TOC graphic

C

N

67LR

Nucleus Cav-1

ROS

Caspase

Apoptosis

Migration, Invasion

43


Improving the Anticancer Efficacy of Laminin ... - ACS Publications

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