Hyaluronic Acid Stabilized Iodine-Containing Nanoparticles with Au

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Hyaluronic acid stabled iodine-containing nanoparticles with Au nanoshell coating for X-ray CT imaging and photothermal therapy of tumors Xinghua Liu, Chunhui Gao, Junheng Gu, Yunfang Jiang, Xinlin Yang, Shao-Yong Li, Wei Gao, Tong An, Hong-Quan Duan, Jing-Wei Fu, Yinsong Wang, and Xiaoying Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11918 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on October 2, 2016

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ACS Applied Materials & Interfaces

Hyaluronic acid stabled iodine-containing nanoparticles with Au nanoshell coating for X-ray CT imaging and photothermal therapy of tumors Xinghua Liu,† Chunhui Gao, † Junheng Gu,‡ Yunfang Jiang,† Xinlin Yang,§ Shaoyong Li,† Wei Gao, † Tong An,† Hongquan Duan, † Jingwei Fu, † Yinsong Wang,*,† Xiaoying Yang*,† †

Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics

(Theranostics), School of Pharmacy, Basic Medical Research Center, Tianjin Medical University, Tianjin 300070, People’s Republic of China ‡

Tianjin Chest Hospital, Tianjin 300051, People’s Republic of China

§

Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry;

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) Nankai University, Tianjin 300071, People’s Republic of China

Xinghua Liu, Chunhui Gao and Junheng Gu contributed equally to this work. * Corresponding authors. No. 22 Qixiangtai Road, Heping District, Tianjin 300070, People’s Republic of China Tel: +86 (22) 8333-6658; Fax: +86 (22) 8333-6690 E-mail addresses: [email protected] (Y. Wang); [email protected] (X. Yang)

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ABSTRACT: In recent years, considerable efforts have been made to the development of multifunctional nanoparticles with diagnosis and therapy functions. To achieve the enhanced CT imaging and photothermal therapy on the tumor, we employed the iodinated nanoparticles as template to construct Au nanoshell structure and demonstrated a facile but effective approach to synthesize the biocompatible and well dispersed multifunctional nanoparticles by coating iodinated nanoparticles with Au nanoshell and the subsequent surface modification of hyaluronic acid. The resultant poly (2-methacryl (3-amide-2, 4, 6-triiodobenzoic acid))/polyethyleneimine/Au nanoshell/hyaluronic acid (PMATIB/PEI/Au nanoshell/HA) nanoparticles had relatively high X-ray attenuation coefficient and photothermal efficiency. After intravenous injection into MCF-7 tumor-bearing mice, PMATIB/PEI/Au nanoshell/HA nanoparticles were efficiently accumulated in the tumor, remarkably enhanced the tumor CT imaging, and selectively ablated the tumor through the thermal treated lesions under the NIR irradiation. Thus, PMATIB/PEI/Au nanoshell/HA nanoparticles displayed a great potential for CT diagnosis and CT guided, focused photothermal tumor therapy.

KEYWORDS: gold nanoshell, iodine-containing nanoparticles, computed tomography, contrast agents, photothermal therapy


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INTRODUCTION Gold (Au) nanostructures exhibit excellent optical and electronic properties, good biocompatibility and in vivo stability, and the easiness of surface modification, thus allowing their use in biological and medical applications. Some special gold nanostructures such as nanoshells, nanocages and nanorods, etc. show the unique surface plasmon resonance (SPR) and have the strong absorption in near-infrared (NIR) region, where the absorption of human tissues is minimal and penetration is optimal. Therefore, these Au nanostructures have great potential for applications in cancer photothermal therapy. In addition, Au nanostructures have been intensively studied as the contrast agents for in vivo X-ray computed tomography (CT) imaging due to its high atomic number (Z=79) and k-edge value (80.7 keV). CT is one of the most powerful non-invasive clinical imaging modalities in modern medicine. Contrast agents are often employed to increase the sensitivity of CT imaging. Currently, most clinically used CT contrast agents are water-soluble iodinated compounds, which are generally derived from 1,3,5-triiodobenzene. However, these small molecular iodinated compounds can be rapidly excreted through the kidney and often exhibit very short blood circulation times.1 With the fast development of nanotechnology, the nanoparticles with high X-ray attenuation coefficients will be the promising candidates for multi-functionalized CT contrast agents. The main purpose of these nanomaterials is to locally increase iodine concentrations, thus resulting in higher local contrast compared with conventional water-soluble CT contrast agents through the enhanced permeation and retention (EPR) effect of the certain sized nanoparticles at the tumor region. During the last decade, iodinated nanoparticles have attracted

intensive

interests,

including

iodine-containing

micellar

nanoemulsion,2,3

iodine-containing polymeric contrast agents,4,5 iodine-containing dendrimeric contrast agents,6 iodine-containing liposomal contrast agents7 and iodine-containing lipoproteins.8 Besides iodine, the nanoparticles based on heavy atoms such as gold, lanthanides, and 3 ACS Paragon Plus Environment


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tantalum have been also investigated as more efficient CT contrast agents. Among them, the gold nanoparticles have been researched extensively due to their synthetic control over size, shape and surface-coating, their high biocompatibility and unique physical properties.9-10 Peng et al. reported a facile approach to prepare dendrimer nanoparticles containing Au and iodine simultaneously and demonstrated that this nanosystem significantly enhanced the X-ray attenuation property of nanoparticles.11 The nanoparticles with both diagnostic and therapeutic functions have been emerging as the next generation platform in the field of nanomedicine. Their capability to simultaneously diagnose and treat cancers, and evaluate therapeutic efficacy may provide efficient combination strategy and synergistic effects in cancer diagnosis and therapy. For example, combining imaging and photothermal therapy may accurately locate the tumor position and then ablate tumor tissue with laser irradiation. Au nanoshells have shown considerable efficacy for tumor ablation using NIR light in many recent research12-15. Au nanoshell has a spherical dielectric nanocore particle surrounded by a thin layer of Au, which can not only ablate tumor under NIR laser irradiating but also be used as CT contrast agent due to its high X-ray attenuation effect. Therefore, Au nanoshell is being explored in multiple clinical trials and shows potential to be a promising next generation candidate for X-ray contrast materials and cancer photothermal therapy.16-19 One of the major disadvantages of Au nanoshells in biomedical applications is their stability in solution due to their metal surface and high density. As an extremely inert material, Au nanopaticles can be surface-modified with biocompatible polymers such as polyethylene glycol,20 oligonucleotides21 and dextran22 to enhance their stability and biocompatibility. Hyaluronic acid (HA) is a highly water-soluble polysaccharide and

composed

of

repeated

disaccharide

units

of

D-glucuronic

acid

and

D-N-acetylglucosamine, linked via alternating β-1,4 and β-1,3 glycosidic bonds. Thus, the surface modification of HA will be favor to enhancing the stability of nanoparticles in the 4 ACS Paragon Plus Environment

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aqueous solution. Futhermore, some investigations have reported that HA is a specific ligand for CD44 that is often over-expressed on tumor cells and the receptors for hyaluronan-mediated motility (RHAMM).23,24 Thereby, HA has been focused on recently to be used as a carrier material for tumor-targeted delivery of anticancer drugs and imaging contrast agents. Au nanoshells were generally synthesized using a template-mediated method. The template may be various nanoparticles, including silica nanospheres, polymer nanospheres and Fe3O4 nanoparticles.25-27 In order to achieve the highest CT value, we employed the iodinated nanoparticles as template to construct Au nanoshell structure. Herein, we demonstrated a facile but effective approach to synthesize the biocompatible and well-dispersed multifunctional particles by coating iodinated nanoparticles with Au nanoshell and the subsequent surface modification of HA.

EXPERIMENTAL SECTION Chemicals. 3-Amino-2,4,6-triiodobenzoic acid was purchased from Alfa Aesar. Methacryloyl chloride, dimethyl sulfoxide-d6 (DMSO-d6), sodium borohydride (NaBH4) and branched polyethyleneimine (PEI, average Mw = 10,000) were purchased from Heowns Biochem. Technologies Llc. Hyaluronic acid (HA, Mw = 35,000) were purchased from Bloomage Freda Biopharm Co. Ltd. N,N-Dimethylacetamide (DMA, analytical grade) was bought from Tianjin Kemiou Chemical Reagent Co. Ltd. and dried over molecular sieve. N, N-Methylenebisacrylamide (MBAAm, chemical grade, Tianjin Bodi Chemical Engineering Co.) was recrystallized from acetone. Acetonitrile (AN, analytical reagent) was got from Tianjin Chemical Reagents II Co. Chloroauric acid (HAuCl4·4H2O) was purchased from BOLT (Tianjin) Chemical Trading Co., Ltd. Hydroxylamine hydrochloride and sodium citrate



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were purchased from Tianjin chemical reagent factory. All reagents were used without further purification and the water was purified through a Millipore system. Synthesis of 2-methacryl (3-amide-2, 4, 6-triiodobenzoic acid) (MATIB). 3-Amino-2, 4, 6-triiodobenzoic acid (4 g, 0.0078 mol) was dissolved in 16 mL of DMA, and methacryloylchloride (2.448 mL, 0.023 mol) was then added. The mixture solution was stirred at 50 °C for about 8 h and then transferred into 80 mL water under vigorous stirring. The white solid was obtained by repeated centrifugations. After that, the product was purified by the recrystallization from alcohol and dried in a vacuum at 50 °C with a yield of 75.3%. 1

H NMR (400MHz, DMSO-d6, δ): 1.96 (s, 3H, CH3), 5.56 (s, 1H, olefinic), 5.93 (s, 1H,

olefinic), 8.37(s, 1H, Ar H), 9.94 (s, 1H, CONH), 13.97 (s, 1H, Ar COOH). Preparation of poly(2-methacryl (3-amide-2, 4, 6-triiodobenzoic acid)) (PMATIB) nanoparticles by precipitation polymerization. PMATIB nanoparticles were prepared by the precipitation polymerization method as follows. MBAAm (0.075 g, 0.49 mmol, 14.3 wt% relative to the total monomer), MATIB (0.45 g, 0.77 mmol ) and AIBN (0.01 g, 0.06 mmol, 2 wt% relative to the total monomer and the crosslinker) were dissolved in 72 mL of acetonitrile and 7.2 mL ethanol (10:1, v/v) in a dried 250-mL two-necked flask attaching with a condenser-Allihn type. The reaction mixture was heated to boiling within 30 min. After that, the initially homogeneous reaction mixture gradually became opalescent and then deepened in color as a milky white dispersion. After 40 min of boiling, the reaction was ended and the resultant PMATIB nanoparticles were separated by centrifugation (12,000 rpm, 20 min) and washed at least three times with acetonitrile. Finally, PMATIB nanoparticles were dispersed in Milli-Q water for the subsequent use. Preparation of PMATIB/PEI/AuNPs. The gold nanoparticles (AuNPs) were prepared according to the modified method in the literature25. Briefly, 1 mL of HAuCl4 aqueous solution (1% w/v) was diluted to 200 mL, and then 6 mL of sodium citrate solution (1% w/v) 6 ACS Paragon Plus Environment

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was added under vigorous stirring. Afterwards, 2 mL of sodium citrate solution (1% w/v) containing 0.075% NaBH4 (w/v) was quickly added, thus obtained AuNPs. The AuNPs dispersion was kept stirring for at least 5 min and then stored at 4 °C before use. 1 mL of PMATIB aqueous solution (0.029 mg mL-1) was slowly added to 1 mL of PEI aqueous solution (1 mg mL-1) and the mixture was stirred for 30 min, thus positively charged PEI was deposited onto the surface of PMATIB with negative charges. The obtained PMATIB/PEI nanoparticles were washed at least three times with water and redispersed in 1 mL of water. After that, 1 mL of PMATIB/PEI nanoparticles solution was added slowly to 100 mL AuNPs dispersion and the mixture solution was vigorously stirred for 12 h. During this period, AuNPs were adsorbed to PMATIB/PEI nanoparticles, and afterwards the excess AuNPs were removed by washing with water. Preparation of Au nanoshell coated PMATIB nanoparticles with HA modification (PMATIB/PEI/Au nanoshell/HA). The Au nanoshells outside the PMATIB nanoparticles were formed by the reduction of HAuCl4 around PMATIB/PEI/AuNPs. Briefly, 0.6 mL sodium citrate solution (1% w/v) and 0.353 mL of HAuCl4 aqueous solution (1%, w/v) were added to 60 mL PMATIB/PEI/AuNPs (0.048 µg mL-1) under stirring and ultrasonication. Afterwards, hydroxylamine solution (80 mM, 0.3 mL) was added dropwise and the mixture solution was stirred under the ultrasonication for 30 min to realize the reduction of HAuCl4. Next, 50 mL of hyaluronic acid (0.2 mg mL-1) was added dropwise and reacted for 6 h at room temperature. The mixture solution was centrifuged and the precipitate was then washed with water for at least three times to obtain PMATIB/PEI/Au nanoshell/HA nanoparticles. In addition, poly (methacrylic acid) (PMAA)/PEI/Au nanoshell/HA nanoparticles were prepared as the control by the same method. Characterization techniques. The proton nuclear magnetic resonance (1H-NMR) spectrum of MATIB was performed using a Brucker AV 400 WB instrument (Brucker AV 7 ACS Paragon Plus Environment


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400). The morphologies of nanoparticles at every step were observed by transmission electron microscope (TEM) using a Hitachi, HT7700 microscope. Energy-dispersive X-ray spectroscopy (EDX) and EDX mapping of nanoparticles were determined by TEM using a FEI, Tecnai G2 F20 microscope. The sizes and size distributions of nanoparticles were measured in water using a laser scattering spectrometer (BECKMAN COULTER, DelsaTM Nano C). The hydrodynamic diameters (Dh) and the polydispersity indexes of nanoparticles were analyzed by the cumulant method. Both of Au and I contents of the nanoparticles were determined by inductively coupled plasma mass spectrometry (ICP-MS) (Thermo Electron Corporation, X7). Fourier translation infrared (FT-IR) spectra were obtained by a Bruker Tensor 27 spectrometer using potassium bromide pellet and the diffuse reflectance spectra were scanned over the range of 400–4000 cm-1. UV-vis-NIR spectra were recorded on an ultraviolet-visible near IR spectrophotometer (JASCO, V-570). Evaluation of colloidal stability of PMATIB/PEI/Au nanoshell/HA nanoparticles. To further evaluate the colloidal stability of PMATIB/PEI/Au nanoshell/HA nanoparticles, the size and size distribution of PMATIB/PEI/Au nanoshell/HA nanoparticles in phosphate buffer saline (PBS, pH 7.4) were determined during a 6-day storage period using the dynamic laser scattering method. The colloidal stability of PMATIB/PEI/Au nanoshell nanoparticles without surface-modification of HA was also evaluated meanwhile. Investigation of photothermal effect of PMATIB/PEI/Au nanoshell/HA induced by NIR laser irradiation. 1 mL of PMATIB/PEI/Au nanoshell/HA nanoparticle solutions with particle concentrations ranged from 0 to 200 µg mL-1 were irradiated using an NIR laser (808 nm, 1.4 W cm-2) for about 20 min until the temperatures reached plateaus. The temperatures of PMATIB/PEI/Au nanoshell/HA nanoparticle solutions were measured using a digital thermometer every one minute. 8 ACS Paragon Plus Environment

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Morphological observation of cellular uptake. The cellular internalizations of PMATIB/PEI/Au nanoshell/HA nanoparticles in MCF-7 cells and human umbilical vein endothelial cells (HUVECs) were observed by TEM. Briefly, MCF-7 cells and HUVECs (1×106 cells per culture flask) were incubated in 5 mL DMEM for 24 h. Afterwards, the culture media were replaced with 5 mL fresh culture media containing PMATIB/PEI/Au nanoshell/HA nanoparticles at Au concentration of 25 µg mL-1, and the cells were further cultured for 4 h. After washing with PBS three times, the cells were collected by centrifugation at 1000 rpm for 3 min and then fixed with 2.5% glutaraldehyde solution. Thus obtained samples were dehydrated in a graded ethanol series followed by propylene oxide, embedded in EPSM812, sectioned using a Diatome diamond knife (Bienne, Switzerland) on a Leica EM UC6 (Vienna, Austria), and finally observed by TEM. Cytotoxicity assay. The cytotoxicity of PMATIB/PEI/Au nanoshell/HA nanoparticles was evaluated in human breast cancer MCF-7 cells by MTT assay. MCF-7 cells were seeded into 96-well plates with cell density of 8×103 per well and incubated for 24 h at 37 °C to allow the cells to attachment. After that, the culture media were replaced with serum-free media containing PMATIB/PEI/Au nanoshell/HA nanoparticles at Au concentrations of 0, 12.5, 25, 37.5, and 50 µg mL-1, respectively, and the cells were then incubated for 4 h. Next, some of these cells were irradiated by an NIR laser irradiation (808 nm, 1.4 W cm-2) for 10 min and incubated for another 18 h. Finally, the viabilities of MCF-7 cells with and without laser irradiation were determined by MTT assay. Measurements of CT values in vitro and CT images of PMATIB/PEI/Au nanoshell/HA in vivo. The CT values of PMATIB/PEI/Au nanoshell/HA nanoparticles with different I concentrations or Au concentrations were measured in vitro. PMAA/PEI/Au nanoshell/HA nanoparticles without I, PMATIB nanoparticles and iohexol were used as the



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controls. All of CT images were acquired using a PHILIP MX-16 SLICE clinical system with 90 kV, 34 mAs and a slice interval of 0.75 mm and SW of 1.5 mm. In vivo bioimaging, the nude mice bearing human breast MCF-7 tumors were provided by Tianjin Saierbio Technology Incorporation. The mouse was intravenously injected with PMATIB/PEI/Au nanoshell/HA suspension (15.2 mg Au mL-1, 300 µL) and scanned using a PHILIP MX-16 SLICE clinical system at 1 h, 6 h and 24 h after administration. The mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (150 µL) before performing CT scans each time. All animal experiments were performed in accordance with the protocols approved by Institutional Animal Care and Use Committee of Tianjin Medical University (Tianjin, China). In vivo tumor photothermal ablation and photothermal imaging. The MCF-7 tumor-bearing mice were randomly divided into four groups (five mice each group), respectively receiving once treatments of normal saline as the controls, PMATIB/PEI/Au nanoshell/HA

nanoparticles,

laser

irradiation,

and

PMATIB/PEI/Au

nanoshell/HA

nanoparticles combined with laser irradiation. 300 µL of PMATIB/PEI/Au nanoshell/HA nanoparticle dispersion (15.2 mg Au mL-1) was injected via tail vein. The 808-nm laser irradiation was carried out locally on the tumor at an out power 3 W cm-2 for 5 min at 6 h post administration of PMATIB/PEI/Au nanoshell/HA nanoparticles. After treatments, the tumor dimensions in the four groups were determined everyday using a caliper and the tumor volumes were calculated according to the following formula: V = ab2/2 (a: the length; b: the width). The relative tumor volumes were also calculated by the ratio of V/V0, where V0 was the original tumor volume before the treatment. Meanwhile, the survival curves of these mice after various treatments were also monitored. In addition, the whole-body infrared thermal images of the mice receiving the combined treatment of PMATIB/PEI/Au nanoshell/HA nanoparticles and laser irradiation were detected using an infrared camera (FLIR SC660) at 0 10 ACS Paragon Plus Environment

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min, 1 min and 3 min during the laser irradiation, and the tumor-bearing mouse with injection of normal saline was used as the control. When the treatments were completed, the control mice and the treated mice with laser irradiation at 6 h after injection of PMATIB/PEI/Au nanoshell/HA were euthanized, and their tumors were removed for the following pathological examination. In vivo toxicity evaluation. The normal mice were randomly divided into two group (five mice each group) and injected respectively with normal saline (the control) and PMATIB/PEI/Au nanoshell/HA nanoparticles (15.2 mg Au mL-1, 300 µL). To evaluate the in vivo toxicity of PMATIB/PEI/Au nanoshell/HA nanoparticles, these mice were euthanized at 30 d after administration, and their main organs including the heart, liver, spleen, lung, and kidney were then removed for the following pathological examination. Pathological examination. The above tumors and organs were fixed with 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) according to the standard procedure. After that, the stained slides were observed and imaged under a fluorescence microscope (IX71, Olympus, Japan).

RESULTS AND DISCUSSION The preparation route is shown in Scheme 1. Briefly, the iodinated nanoparticles (PMATIB) were

first

prepared

by

(3-Amide-2,4,6-triiodobenzoic

the acid)

precipitation (MATIB)

polymerization

using

N,

of

2-methacryl

N-methylenebisacrylamide

(MBAAm) as a crosslinker. MATIB was synthesized via amide linkage between methacryloyl chloride and 3-amino-2,4,6-triiodobenzoic acid, a procursor compound of most currently clinical CT contrast agent, and its NMR spectrum is shown in Figure S1(see the supporting information). Next, PMATIB nanoparticles were modified orderly with PEI and ultra-fine Au nanoparticles

(AuNPs

Hyaluronic Acid Stabilized Iodine-Containing Nanoparticles with Au

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