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Indocyanine Green-holo-Transferrin Nanoassemblies for TumorTargeted Dual-Modal Imaging and Photothermal Therapy of Glioma Mingting Zhu, Zonghai Sheng, Yali Jia, Dehong Hu, Xin Liu, Xianyuan Xia, Chengbo Liu, Pan Wang, Xiaobing Wang, and Hairong Zheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14076 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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ACS Applied Materials & Interfaces

Indocyanine Green-holo-Transferrin Nanoassemblies for Tumor-Targeted Dual-Modal Imaging and Photothermal Therapy of Glioma Mingting Zhu,†‡# Zonghai Sheng,‡# Yali Jia,†‡ Dehong Hu,‡ Xin Liu,‡ Xianyuan Xia,§ Chengbo Liu,§ Pan Wang,† Xiaobing Wang,†‡* and Hairong Zheng‡* †

Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry,

Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an 710119, China. ‡

Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of

Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. §

Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen

Key Laboratory for Molecular Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. E-mail: [email protected] (Xiaobing Wang); [email protected] (Hairong Zheng) #

Mingting Zhu and Zonghai Sheng contributed equally to this work.

KEYWORDS: holo-Transferrin, Indocyanine green, fluorescence imaging, photoacoustic imaging, photothermal therapy, glioma

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ABSTRACT: Active-targeted cancer imaging and therapy of glioma has attracted much attention in theranostic nanomedicine. As a promising tumor-targeting ligand, holo-transferrin (holo-Tf) has been applied for enhancing delivery of nanotheranostics. However, holo-Tf-based nanoassemblies for active targeting mediated multi-modal imaging and therapeutics have not been previously reported. Here, we develop a one-step method for the preparation of holo-Tf-indocyanine green (holo-Tf-ICG) nanoassemblies for fluorescence (FL) and photoacoustic (PA) dual-modal imaging and photothermal therapy (PTT) of glioma. The nanoassemblies are formed by hydrophobic interaction and hydrogen bonds between holo-Tf and ICG, which exhibit excellent active tumor-targeting and high biocompability. The brain tumor with highly expressed Tf receptor can be clearly observed with holo-Tf-ICG nanoassemblies base on FL and PA dual-modal imaging in subcutaneous and orthotopic glioma models. Under the near-infrared laser irradiation, the holo-Tf-ICG nanoassemblies accumulated in tumor regions can efficiently convert laser energy into hyperthermia for tumor ablation. The novel theranostic nanoplatform holds great promise for precision diagnosis and treatment of glioma.


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1. INTRODUCTION Cancer has been a great threat to human health.1 Generally, cancer treatment mainly includes surgery, chemotherapy and radiotherapy. Nevertheless, surgery can’t remove all the tumor cells in the body, radiotherapy and chemotherapy suffer from unsatisfactory tumor targeting and severe systemic side effects.2 Therefore, in cancer theranostics, precision diagnosis and efficient treatment are extremely important to obtain the optimal therapeutic effect. For decades, the emerging nanotechnology gives new hope to cancer theranostics, especially using multifunctional nanomaterials with highly integrated features.3-8 Currently, several nanomaterials have been developed and translated in preclinical and clinical applications.9,10 Among them, the natural protein-based nanoplatform such as albumin and ferritin, has gained much focus.11-15 For instance, Liu and co-workers reported albumin-based nanoplatform for multimodal imaging and combined therapy.16 Albumin could also be utilized to coat various types of nanoparticles to enhance their biocompatibility.17 Although albumin shows some level of tumor targeting ability by Gp60 receptor-mediated endocytosis,18 but the tumor-targeting of individual albumin-dye/drug complexes with small sizes is usually limited by relatively low stability (exchange of molecular binding with endogenous albumin


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and other proteins in the blood).16 Ferritin (Fn) is one kind of protein that stores iron in the body tissue. Xie et al.19 reported a RGD4C-modified Fn nanoparticles (RFRT) that could exhibit fluorescence (FL) imaging and efficient photodynamic therapy. Chen et al. synthesized a near-infrared (NIR) dye (IR820)-loaded Fn nanocages (DFRT) by acid dissociation method for FL and photoacoustic (PA) imaging guided-photothermal therapy (PTT).20 Although these studies have achieved some exciting results, design and preparation of all-in-one theranostic nanoplaform is still a challenging concern. Transferrin (Tf), the transport protein in biological metabolism, has a variety of applications (Table 1).21-30 It can specifically binds to the transferrin receptor (TfR) that highly expressed in many tumor cells via ligand-receptor interaction.3133

As one of the Tf family, holo-Tf contains two iron atoms and exhibits the

highest affinity with TfR than that of apo-Tf (iron-free Tf), mono iron Tf (contains one iron atom).34 Therefore, holo-Tf can be generally applied as a specific ligand for tumor-targeted imaging and therapy. Recently, Han et al.22 reported holo-Tfbased biomineralized gadolinium-holo-Tf (Gd@Tf) nanoparticles with systematic clearance for active targeting magnetic resonance imaging. Liu et al.35 prepared a carbocyanine

dyes-gadolinium-diethyle-netria-minepentaaceticacid-holo-Tf


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(Cy5.5-Tf-DTPA-Gd) to enhance the diagnosis of non-small cell lung cancer. The previous studies pave a way for design and synthesis of holo-Tf-based nanoparticles for cancer theranostics. In this study, we develop a molecular assembly-based strategy for the preparation of holo-Tf theranostic nanoplatform for FL and PA dual-modal imaging and PTT of glioma. The assembled process relies on hydrophobic interaction and hydrogen bonds between holo-Tf and indocyanine green (ICG), an FDA-approved NIR FL dye. ICG has the function of dual FL and PA imaging, which can also be an effective sensitizer to stimulate heat for PTT through the 808 nm laser.36,37 However, the application of free ICG is subjected to many limitations, involving water instability, concentration-dependent aggregation, and quick clearance in vivo.21,38,39 In order to overcome these disadvantages, ICG was encapsulated into holo-Tf in the present study with a one-step green method. The holo-Tf-ICG nanoassemblies possess the following characteristics: (I) effective active-targeting, (II) available biosecurity, (III) efficient cancer theranostics (Scheme 1). With these features, the highly integrated nanoplatform is expected to be applied for future clinical translation.


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Table 1. The application of different kinds of Tf in a variety of materials.


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Scheme 1. Schematic illustration of holo-Tf-ICG nanoassemblies for in vivo dual-modal imaging and photothermal treatment.

2. MATERIALS AND METHODS 2.1. Materials. ICG and holo-Tf were obtained from Sigma-Aldrich. Cell Counting Kit (CCK-8), 4, 6-diamidino-2-phenylindole (DAPI) were provided by Beyotime Biotechnology. Calcein-AM and Propidium Iodide (PI) were purchased


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from YEASEN (Shanghai, China). Fetal bovine serum (FBS), trypsin-EDTA penicillin-streptomycin, phosphate-buffered saline (PBS, pH = 7.4), DMEM medium, F12K medium, B-27™ supplement and N-2 supplement were obtained from Gibco LifeTechnologies (AG, Switzerland). Human EGF and human FGFbasic were purchased from Pepro Tech (USA). Anti-TfR antibody (ab84036) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) were purchased from Abcam (Cambridge, UK). Human glioma U87 cell line and mouse brain microvascular endothelial (bEnd.3) cell line were obtained from Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. Female athymic nude mice (Balb/c) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. 2.2.

Synthesis

of

holo-Tf–ICG

Nanoassemblies.

The

holo-Tf-ICG

nanoassemblies were prepared using a self-assembly method. Briefly, 3 mg holoTf and 0.9 mg ICG were dissolved in 2 mL water and reacted under vigorous stirring at room temperature for 12 h. Then, the mixed solution was dialyzed with water for 36 h to remove free ICG. Finally, the prepared holo-Tf-ICG nanoassemblies were stored at 4 °C. All the experiments were conducted in the dark.


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2.3. Molecular Docking. The interactional model between ICG and holo-Tf was established using the DOCK package on SGI O2 workstation, and the molecular simulation was carried out. The crystal structure of holo-Tf was obtained from the PDB protein crystal database, encoded 3V83. ChemDraw tool was used to construct the molecular structure of ICG, and then the experimental molecular model was optimized based on the molecular mechanics MM2 force field. Using AutoDock Toolkit (ADT) to optimize the receptor and ligand. 2.4. Characterization of holo-Tf-ICG Nanoassemblies. The size of holo-Tf–ICG nanoassemblies was determined using a JEM 1200EX transmission electron microscope (TEM). The hydrodynamic size and zeta potential were determined using a Malvern Zetasizer (Nano series ZS, UK). The absorption spectrum of free ICG and holo-Tf-ICG were measured by an UV−vis-NIR spectrometer and the FL spectra were detected by a FL spectrophotometer. The circular dichroism (CD) spectrum were recorded on a Model Jasco J-715 CD spectrometer at ambient temperature. 2.5. Cell Cultures. U87 cells and bEnd.3 cells were incubated in DMEM medium supplemented with 1% penicillin–streptomycin (v/v) and 10% (v/v) FBS at 37 °C under a humidified atmosphere with 5% CO2.


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2.6. Cytotoxicity of Free ICG and holo-Tf–ICG Nanoassemblies. U87 cells were seeded in a 96-well plate with 104 cells/well and incubated for 24 h. Then, the cells were incubated in opti-MEM (Gibco) with different ICG doses in either free ICG form or holo-Tf-ICG nanoassemblies for 4 h. After that, adding the CCK-8 solution and incubated for 4 h in a cell incubator. Finally, a multi-functional microplate reader was utilized to detect the absorbance of the solution in each well at 450 nm. There were five repetitions in each group. 2.7. holo-Tf Receptor Expression Levels of U87 and bEnd.3 Cells. U87 cells were seeded in an eight-chamber slide with 2 × 104 cells per well and cultured for 24 h. After washing with PBS for three times, fixed with 4% paraformaldehyde and added anti-TfR antibody at 4 ℃ overnight, the goat anti-rabbit IgG H&L was applied and incubated for 1 h at 37 ℃ , then counterstained with DAPI and observed under a laser confocal microscopy. 2.8. The Active Targeting Ability of holo-Tf-ICG. U87 and bEnd.3 cells (2 × 104 cells/well) were seeded onto an eight-chamber slide and incubated for 24 h at 37 °C. 20 µg/mL ICG in either holo-Tf-ICG or free ICG were added to the solution and incubated for 4 h, respectively. After washing with PBS for three times, cells were fixed in 4% paraformaldehyde at room temperature for 10 min. Then, the


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slides were mounted with DAPI and imaged by a laser scanning confocal fluorescence microscope. For cellular uptake determination, the cells (2 × 104 cells/well) were seeded in 24-well plates and cultured with 20 µg/mL ICG in either holo-Tf-ICG nanoassemblies or free ICG form. After incubated for 4 h, rinsed, trypsinized and resuspended cells, the uptake of ICG were analyzed quantitatively by flow cytometer (BD Accuir C6, USA). 2.9. Tumor Spheroid Penetration. We further evaluated the tumor-penetrating ability of holo-Tf-ICG. U87 cells (104 cells/well) were seeded in a 24-well plate which has the ultra-low attachment surface (Corning, USA), then cells were incubated using serum-free F12K medium containing growth factors (EGF, FGF, B-27TM, N-2). The tumor spheroids were formed after one week, following further incubation for 4 h with free ICG, holo-Tf-ICG at an ICG concentration of 20 µg/mL. The tumor spheroids were then rinsed three times and observed under a laser scanning confocal microscope. The tumor spheroids were scanned in multiple layers to obtain the FL image, and the interval between consecutive slides was 20 µm 2.10. In Vitro PTT. The cell viability assays was performed by a standard CCK-8 assay. U87 cells (104 cells/well) was seeded on 96-well plate and incubated for 24


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h at 37 °C. The cells were treated with free ICG and holo-Tf-ICG (20 µg/mL ICG) and incubated for 4 h at 37 °C. After incubation, the cells were washed three times and irradiated different times (0, 2, 4, 6, 8, and 10 min) with a 808 nm laser (an NIR laser source was from Beijing Laserwave Optoelectronics Tech. Co., Ltd.) at 0.8 W/cm2. On the one hand, the cells were treated with different concentrations of free ICG and holo-Tf-ICG (ICG concentration: 0, 1, 5, 10, 15, 20 µg/mL) and incubated for 4 h at 37 °C. After incubation, the cells were washed and irradiated 10 min with a 808 nm laser at 0.8 W/cm2. Calcein AM/PI was also conducted for cell viability assay. Briefly, U87 cells (104 cells/well) were seeded on 96-well plates and incubated for 24 h at 37 °C. Cell culture media were replaced by fresh culture media containing free ICG/holo-Tf-ICG (20 µg/mL ICG). After incubation for 4 h, cells were irradiated by a 808 nm laser (0.8 W/ cm2) for 10 min and then washed with PBS three times. After incubation for another 4 h, the cells were stained with calcein AM/PI, and the cells were imaged by Leica DMI6000 inverted microscope. 2.11. In vivo FL and PA Imaging. All the animal experiments were approved by the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences Animal Care and Use Committee. Balb/c nude mice (6-8 weeks) with


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subcutaneous-tumors in the lower right side of the back area and orthotopic-tumors in the right front of the brain were used as the animal models, respectively. The orthotopic-tumor growth was monitored by magnetic resonance imaging after being implanted 10 days. The imaging experiments were carried out when the subcutaneous-tumors grew to 60 mm3, and the orthotopic-tumors grew to 2-3 mm in diameter. The holo-Tf-ICG nanoassemblies or free ICG (1 mg ICG/kg) were intravenously injected into mice (n = 5 in each group) through tail-vein, and then FL images at different time points were acquired on an in vivo FL imaging system (IVIS Spectrum, PerkinElmer). In vivo PA imaging of subcutaneous-tumor model was executed with preclinical PA computerized tomography scanner (Endra Nexus 128, Ann Arbor, MI). The excitation wavelength was 780 nm. Besides, we used a tunable pulsed OPO laser (Vibrant 355 II HE) with a repetition rate of 10 Hz and a pulse width of 5 ns to illuminate the PA imaging for orthotopic-tumor model through a custom-made light delivery system. The excitation wavelength was also 780 nm. 2.12. In Vivo Thermal Imaging. When the subcutaneous-tumors reached 60 mm3, holo-Tf-ICG nanoassemblies (1 mg ICG/kg) or PBS was intravenously injected into the nude mice bearing U87 tumors. Thermal imaging was presented by an


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infrared thermal imaging camera (Ti27, Fluke, USA) when the tumors were subjected to 808 nm laser with the power density of 0.8 W/cm2. 2.13. In Vivo PTT. 100 µL PBS, holo-Tf-ICG solution or free ICG (1 mg ICG/kg) were intravenously injected into the nude mice with subcutaneous-tumors (n = 5 in each group). At 24 h after injection, the tumors were exposed to a NIR laser of 0.8 W/cm2 for 5 min. The tumor photos at different time points before and after injection of holo-Tf-ICG nanoassemblies were recorded to monitor the volume changes of tumors. The tumor inhibition ratio, the tumor sizes and mouse weight were measured at different time points. 2.14. H&E Staining. For H&E staining, briefly, 8 µm slides of organs were fixed with 4% paraformaldehyde solution, after that dehydrated, performed H&E staining. 3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of holo-Tf-ICG Nanoassemblies. The onestep prepared process of holo-Tf-ICG nanoassemblies is shown in Figure 1a. After facile mixing and dialysis procedures, the ICG loaded holo-Tf was obtained. The interaction of holo-Tf and ICG was evaluated by molecular docking to identify their active binding sites and energy (Figure 1b, 1c). The crystal structure of holo-


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Tf was taken from PDB crystal database (No. 3v83). ICG molecules can bind on the surface active pockets of holo-Tf through the hydrophobic interaction, and their binding energy was measured to be ~18.7 kcal/mol (Table S1), revealing a good affinity.40 The three-dimensional structure of holo-Tf indicates that the hydrogen bond was formed by interaction between the oxygen atoms of ICG and NH2 group of holo-Tf. The hydrogen bond energy was estimated to be 100 (Table S1), indicating efficient binding of ICG molecules and holo-Tf. It is estimated that hydrophobic interaction and hydrogen bonds are the general driving force for the formation of holo-Tf-ICG nanoassemblies. Such interaction behavior has been reported in others’ experimental systems. Moosavi-Movahedi et al. verified that hydrophobic forces play an important role in the electron transfer and protein folding mediated by the molten globule state of cytochrome c.41 Researchers investigated the interaction between different proteins and small molecules 42-48 by molecular modeling, fluorescence quenching, resonance light scattering, circular dichroism, density functional theory, etc., such as interaction behavior between tamoxifen/lomefloxacin and human holo-Tf,43,44 they found the hydrophobic interactions played a vital roles in distinct complex formation. In addition, electrostatic interactions and the formation of hydrogen bonds also performed a


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certain effect. These reports indirectly support our viewpoint that the nanoassembly of holo-Tf-ICG is dependent on the hydrophobic interaction and the presence of hydrogen bonds.

Figure 1. The molecular model of interaction between holo-Tf and ICG. (a) Schematic illustration shows the synthetic process of holo-Tf-ICG nanoassemblies. (b) Crystal structure of holo-Tf-ICG complex (The image below shows amplification of the red circle above, white region represents the hydrophobic area). (c) Three-dimensional pattern of holo-Tf-ICG complex (The image below shows the amplification in the red circle area, the black dashed lines in the blue circles indicate the formation of hydrogen bonds).


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The assembled process of ICG and holo-Tf was further optimized by changing the mass ratio of ICG to holo-Tf (Table 2). The ICG loading efficacy increased with increasing the mass ratio of ICG to holo-Tf from 1:20 to 1:2. The optimal holo-Tf-ICG nanoassemblies (ICG: holo-Tf = 3:10) were obtained with drug efficiency of 8.61%. It could be seen from Figure S1 that the additional ICG would not be encapsulated when it was above 30 %, this is in agreement with the results in Table 2. TEM images indicate that the nanoassemblies present a spherical morphology with particle size around 10 nm (Figure 2b), which is slightly larger than that of pure holo-Tf (~ 9 nm) as shown in Figure 2a, indirectly demonstrating

effective

load

of

ICG

on

holo-Tf.

DLS

measurement

further validates that the hydrodynamic size of holo-Tf increased from 10.5 nm to 12 nm after binding with ICG (Figure 2c, 2d). As shown in Figure 2e, the obtained nanoassemblies exhibit dark green color and excellent colloidal stability after 14 day storage (4 ℃). The absorption spectrum of nanoassemblies is similar to that of free ICG (Figure 2f), however, their FL intensity decreased 73% compared to that of free ICG (Figure 2g), suggesting that sufficient loading of ICG on the surface of holo-Tf can induce its FL self-quenching. CD spectrum of holo-Tf was not obviously influenced by loading of ICG (Figure 2h), revealing that the secondary


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structures of holo-Tf-ICG, including α helix, β sheet and random coil, have no obvious changes compared with pure holo-Tf. This bionic synthesis method is green, mild and would not interfere with the inherent geometry of holo-Tf. Table 2. Effects of the mass ratio of ICG to holo-Tf on the size, zeta potential, drug loading and encapsulation efficiency of holo-Tf-ICG nanoassemblies.


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Figure 2. Characterization and physiochemical properties of holo-Tf-ICG nanoassemblies. Representative TEM images of pure holo-Tf (a) and holo-Tf-ICG nanoassemblies (b). The size distribution of pure holo-Tf (c) and holo-Tf-ICG nanoassemblies (d) using DLS. (e) The particle size change of holo-Tf-ICG nanoassemblies during 14 days’ storage. The inset shows the photographs of holo-Tf-ICG solutions at different time point. (f) UV−vis absorbance spectra of free ICG and holo-Tf-ICG nanoassemblies. (g) FL spectra of free ICG and holo-TfICG nanoassemblies. (h) CD spectra of pure holo-Tf and holo-Tf-ICG nanoassemblies.

3.2. Cellular Uptake of holo-Tf-ICG. TfR is a transmembrane protein that specifically binds with Tf. TfR has a low level of expression in most normal endothelial cells, and is relatively high expression in glioma cells.49 In order to verify the targeting ability of holo-Tf-ICG, the expression levels of TfR in U87 glioma cell line and bEnd.3 brain microvascular endothelial cells line were detected by immunofluorescence experiment (Figure 3a). It is observed that U87 cells presented much higher expression level of TfR than that of bEnd.3 cells. Then, the cellular uptake of holo-Tf-ICG was further investigated by confocal fluorescence microscopy (Figure 3b). Compared to free ICG, holo-Tf-ICG-treated U87 cells showed a stronger red FL than that of bEnd.3 cells. Flow cytometry analysis shows that the FL intensity of holo-Tf-ICG-treated group was 2.4-fold higher than that of free ICG-treated group (Figure 3c), indicating that holo-Tf-ICG


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with excellent active targeting can selectively enhance the cellular uptake of ICG. Generally, nanomedicine with surface decorated with targeting peptide or ligand improved cellular uptake via receptor-ligand mediated endocytosis.50 Herein, we suppose holo-Tf-ICG (holo-Tf with high affinity with TfR51) specially binds with TfR on U87 cell surface, Then, the holo-Tf-ICG-TfR complex is clustered into the recessed sites of the cell surface network protein, and the recess caved with partial membrane shedding into the cell to form endocytosis body, thus facilitating ICG’s specific uptake. Next, the 3D tumor spheroids of U87 cells were developed for further evaluating the penetrative effect of holo-Tf-ICG nanoassemblies. As shown in Figure 3d, nanoassemblies were easier to penetrate into 3D tumor sphere and exhibited stronger FL than that of free ICG, indicating holo-Tf can improve the permeation of ICG through acceptor-receptor interaction. To evaluate the cytotoxicity of nanoassemblies, standard CCK-8 assay was performed (Figure S2). It indicates that the cell viability was over 90% after treatment of U87 cells with different concentrations of holo-Tf-ICG and free ICG, respectively. These results reveal that holo-Tf-ICG with low cytotoxicity can not only improve tumor-


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targeting ability, but also enhance the tumor penetration, which would play an important role in cancer therapy.

Figure 3. Uptake and permeability of holo-Tf-ICG. (a) Immunofluorescence imaging of TfR expression in U87 and bEnd.3 cells. (b) Subcellular localization of ICG in U87 and bEnd.3


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cells after incubation with free ICG and holo-Tf-ICG for 4 h. (c) Mean FL intensities of free ICG and holo-Tf-ICG in U87 and bEnd.3 cells measured by flow cytometry (n = 104 cells). Data are presented as means ± SD from three independent experiments. **p

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