Gold Nanorods-Based Smart Nanoplatforms for Synergic

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Biological and Medical Applications of Materials and Interfaces

Gold Nanorods-Based Smart Nanoplatforms for Synergic Thermotherapy and Chemotherapy of Tumor Metastasis Bin Li, Yiqing Wang, and Jian He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21784 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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ACS Applied Materials & Interfaces

Gold Nanorods-Based Smart Nanoplatforms for Synergic Thermotherapy and Chemotherapy of Tumor Metastasis Bin Li,† Yiqing Wang,*† Jian He*# †Department

of Biomedical Engineering, College of Engineering and Applied Sciences,

Nanjing University, Jiangsu Province 210093, China #

Department of Radiology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing

University Medical School, No. 321 Zhongshan Road, Nanjing 210008, China

KEYWORDS: gold nanorods, chemotherapy, polydopamine, metastasis, photoacoustic imaging

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ABSTRACT: The combination therapy of photothermal therapy (PTT) and chemotherapy as a promising strategy have drawn extensive attention by overcoming limitations of conventional treatments in tumor therapy. Gold nanorods-based nanoplatforms were herein designed by integrating doxorubicin (DOX) and polydopamine coated gold nanorods (GNRs@PDA) for tumor metastasis inhibition and multifunctional drug delivery. The GNRs@PDA-PEG-DOX nanocomplex showed the robust stability and the excellent near-infrared (NIR) photothermal conversion efficiency under laser irradiation. The release of loaded DOX from GNRs@PDAPEG-DOX nanocomposites was improved in tumor microenvironments. Furthermore, the PDA functionalized GNRs nanocomposites were expected to be potential photoacoustic imaging (PAI) agents for imaging-guided tumor therapy. Upon NIR laser irradiation, the efficiency of tumor inhibition of GNRs@PDA-PEG-DOX exhibited greater than the other group in vitro and in vivo, which was confirmed by the immunohistochemistry (IHC) staining, demonstrating a promising strategy for suppression of tumor metastasis and low long-term systemic toxicity. These results illustrated a promising strategy of tailor-made GNRs@PDA-PEG-DOX nanoplatforms for ablation of tumor and suppression of tumor metastasis in clinical application.

1. INTRODUCTION Recently, with an advancing development in the field of conventional tumor therapy strategies including chemotherapy, radiotherapy and surgery, the survival of patients has remarkable improved.1-5 However, metastasis, which is the intrinsic nature of the malignant tumor, remains a challenge in clinical settings.5 In this regard, there is a great need for developing a newly


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efficient tumor therapy strategy to remit and overcome it. Photothermal therapy (PTT) was a noninvasive technology to eliminate tumor, which possessed the outstanding therapeutic outcome and minimal systemic toxicity.6-12 Hence many nanomaterials as PTT agents with near-infrared (NIR) absorbance were designed to convert light to heat at local tumor for photothermal therapy, such as nanosized graphene oxide (NGO),13, 14 metal nanoparticles 15 and organic molecular.16 In these agents, gold nanorods (GNRs) have attracted increasingly attention by exhibiting precise controlled size and morphology, high NIR light absorption and multifunctional nanocarriers.17 Importantly, GNRs as the contrast agent with surface plasmon resonance (SPR)-induced precisely tunable absorption enabled the tumor to visualize in imaging strategies, such as photoacoustic tomography imaging and two-photon luminescence imaging in vitro and in vivo.18-20 Therefore, GNRs-based nanocomplex held promising properties to realize multifunctional nanoplatforms for tumor metastasis inhibition. Despite the satisfactory therapeutic outcome, PTT was still limited by the unavoidably depthdependent of laser irradiation.21 As we all know, chemotherapy as a conventional treatment strategy, always acted a significant role for tumor elimination and metastasis suppression. Therefore, the synergistic therapeutic outcomes of PTT and chemotherapy has been extensively explored, especially for metastasis suppression. Very recently, CuS-doxorubicin (CuS-DOX) nanocapsules as a drug delivery system has been reported to restrain the metastasis by synergic chemotherapy/PTT effects.22 To further improve the therapeutic efficacy, numerous functional materials were investigated by coating GNRs as nanoplatforms, allowing highly chemotherapeutic drug loading.23-27 The



robust polydopamine (PDA) nanospheres which were fabricated by self-polymerization of dopamine under alkaline condition have been reported, holding excellent photothermal conversion efficiency and biocompatibility.28,

29

Meanwhile, benefiting from spontaneous

deposition on virtually any substrates surface, the versatile PDA-coated gold nanocomplex also embodied these intriguing properties. Importantly, the numerous free quinone groups on the surface of PDA layer ensured modification with thiols amines-contained molecules, facilitating the formation of multifunctional nanoplatforms.11, 29, 30 For instance, PDA capped GNRs as nanoplatforms realized the drug carrier system via pH-mediated release to enhance therapy outcome.26 Furthermore, the nanoprobes based on GNRs@PDA core-shell were designed for multimodal imaging and angiogenesis-targeted tumor therapy.10 However, the long-term systemic toxicity and metastasis inhibition property of GNRs@PDA nanoparticles remains to be explored. In this study, we described the fabrication of a robust and multifunctional GNRs@PDA-PEGDOX nanoplatforms to seek synergistic effect for the combination of PTT and chemotherapy, allowing to explore the long-term systemic toxicity and metastasis inhibition. The as-obtained PEGylated GNRs were surrounded by PDA shell for insulating the protein layer and enhancing the biocompatibility. Additionally, GNRs@PDA-based nanoparticles as a vital drug delivery system carried the DOX to decrease the systemic toxicity in vivo for inhibition of primary solid tumor and metastasis. Under the laser irradiation and tumor acidic microenvironment, synergistic therapies exhibited a satisfactory curative effect in vitro and in vivo. Based on these studies in this contribution, PDA capped GNRs nanocomposites could be potential


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nanoplatforms for high-efficiency tumor ablation and suppression of neoplasm metastasis.

2. EXPERIMENTAL SECTION 2.1 Materials

and

animals.

All

experiment

materials

were

purchased

from

commercial corporations. Hydrogen tetrachloroaurate (Ⅲ) tetrahydrate (HAuCl4·4H2O), silver nitrate (AgNO3), dopamine hydrochloride (DA) and sodium borohydride (NaBH4) were brought from Sigma-Aldrich (Shanghai, China). Cetyltrimethylammonium bromide (CTAB), ascorbic acid and sodium oleate (NaOL) were bought from Tokyo Chemical Industry (TCI, Tokyo, Japan). The sodium hydroxide (NaOH) and hydrochloric acid (HCl) were obtained from Shanghai Sinopharm Chemical Reagent Co., Ltd (China). All polymers (mPEG-SH 5000, H2NPEG-SH 5000), doxorubicin hydrochloride (DOX) and tris(hydroxymethyl) aminomethane hydrochloride were purchased from Aladdin (Shanghai, China). The mouse monoclonal Ki67 antibody was purchased from Abcam (Shanghai, China). The Hela cells were obtained from Cell Bank of Shanghai Institutes for Biological Science (Shanghai, China). All mice were brought from the Comparative Medicine Center of Yangzhou University (Yangzhou, China). The ultra-pure water was obtained from Mili-Q deionized water (Millipore, 18.2 MΩ cm-1).

2.2 Instrumentation. The nanoparticle morphology and size directly determined by transmission electron microscopy (TEM, JEM-200CX, Japan). Then, the Shimadzu UV-3600 spectrophotometer (UV-3600, Japan) was utilized to investigate the absorption spectrum of whole nanoparticles. For zeta potential of nanoparticles, the Zetasizer Nano-ZS90 (U.K) was



employed to record the changes of all types of nanoparticles in every step. Afterwards, to investigate the thermostability and photothermal effects of gold nanoparticles, an 808 nm diode laser (Xilong Company, China) was directly utilized in these experiments. The temperature change of all samples was monitored by a thermal camera (Fortic 225-1, Shanghai, China) after exposing in the laser at various times. For cell live or death, all fluorescence images in vitro were recorded by an inverted fluorescence microscope (Olympus, IX73, Japan). For the MTT test, the absorption value of samples was obtained by Infinite® 200 Pro NanoQuant (Tecan) which as a microplate reader. For photoacoustic imaging, the Endra Nexus128 was utilized and made the record. In this work, the concentration of gold was directly evaluated by inductively coupled plasma optical emission spectrometry (ICP-OES, OPTIMA5300DV, PE).

2.3 Synthesis of GNRs. The as-obtained gold nanorods (GNRs) with a NIR longitudinal localized surface plasmon resonance (LSPR) peak were prepared by a seed-mediated method according to the pioneering report.31 Briefly, the gold nanorods (GNRs) growth solution was prepared by adding 5 mL of HAuCl4 (4 mg/mL) to 15 mL of solution (0.064 g NaOL and 0.325 g CTAB). Next, the 0.4 mL of 8 mM AgNO3 and 100 μL of L-ascorbic acid (0.064 M) were injected respectively into growth solution. The GNRs seed solution was prepared by injecting 0.25 mL of HAuCl4 (4 mg/mL) and 5 mL of CTAB solution (0.2 M) into 4.75 mL of H2O. Immediately, 1 mL of 0.017 mmol/ mL fresh NaBH4 solution was injected in one shot. After 2 min vigorous stirring, the gold seed particles were kept undisturbed under room temperature for 30 min. Then the growth solution was injected with 80 μL of gold seed and kept undisturbed


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for 12 hours. The mixture solution was centrifugated at 8500 rpm for 15 min to collect the GNRs.

2.4 Preparation of PEGylated GNRs. For the preparation of PEGylated GNRs, the procedure was employed through previous reports.30 Typically, 0.5 mL of 1 mg/mL mPEG-SH (Mw = 5000 Da) was dropwise added into 1 mL as-synthesized GNRs (0.2 mg/mL) solution and the mixture was stirred for 3 hours. Then the PEGylated GNRs were retrieved through centrifugation and washed twice by deionized water.

2.5 Synthesis of GNRs@PDA, GNRs@PDA-PEG and GNRs@PDA-PEG-DOX. The PEG-modified GNRs were suspended in tris (hydroxymethyl) aminomethane buffer (Tris buffer, 10 mM, pH 8.5) and sonicated for 10 s. The dopamine in deionized water was added into PEGylated GNRs solution with different final concentrations (2 mM and 4 mM). The suspension was mixed and sonicated for 10 min. Under alkaline conditions, the dopamine could self-polymerize and coat directly on the surface of PEGylated GNRs as catechol groups were oxidized to quinones. Finally, the centrifugation was conducted to collect the GNRs with PDAshell nanoparticles (8500 rpm, 15 min). The as-synthesized GNRs@PDA was re-dispersed into deionized water. For the PEG-coated GNRs@PDA, 1 mg of GNRs@PDA was resuspended in 1 mL of HS-PEGNH2 solution (4 mg/mL, Mw 5000). The mixture was injected with sodium hydroxide to adjust the pH value to 12. Then the mixture was stirred and incubated for one night. The centrifugation



was employed to remove free polymer and GNRs were collected. To load the anti-cancer drug doxorubicin (DOX) into GNRs@PDA-PEG nanoparticles, the GNRs@PDA-PEG (30 mg) was resuspended into 3 mL of DOX solution (3.17 mg/mL). The pH value was adjusted to 8.5 and sonicated for 1 min. The mixed solution was kept in the dark room and stirred for another 12 hours. The free DOX was purified via centrifugation and monitored by absorption measurement at 480 nm. The concentration of free DOX was calculated through a standard curve (in Figure S4). The loading efficiency of DOX was evaluated from the equations: Loading efficiency of DOX=(weight of DOX-loaded/ the weight of GNRs@PDA-PEG-DOX) ×100%.

2.6 Characterization of nanocomposites. For the morphology and size of nanocomposites, the transmission electron microscopy (TEM, JEM-200CX, Japan) was utilized. The Shimadzu UV-3600 spectrophotometer (UV-3600, Japan) was used to investigate optical absorption spectra of nanocomposites. The zeta potentials of nanocomposites were recorded by the Malvern Zetasizer Nano-ZS90 (U.K).

2.7 The release of DOX from GNR@PDA-PEG-DOX. To investigate the DOX release profile of GNRs@PDA-PEG-DOX, 1 mg of previously obtained GNRs@PDA-PEG-DOX was resuspended into 1 mL of either pH 5.0 or pH 7.0 solution. The solution was centrifugated at 8500 rpm for 15 min and the supernatant was collected at the fixed period. Meanwhile, the precipitate was re-dispersed into same volume deionized water at each time point. The


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absorption value of free DOX was recorded by UV-vis spectrum. The amount of free DOX was calculated by the standard curve of DOX.

2.8 In vitro photothermal evaluation. To evaluate the photothermal effects of GNRs@PDAPEG, an 808 nm laser irradiation with the power density of 2 W/cm2 was employed. With the addition of GNRs@PDA-PEG nanoparticles solution in a quartz cell, the temperature change of samples at different concentration (20 μg/mL and 100 μg/mL, based on gold ion concentration) under laser irradiation was monitored by a thermal camera for 10 min.

2.9 Evaluation of cell cytotoxicity in vitro. To assess the cytotoxicity in vitro, the Hela cells were cultured in DMEM supplemented with 10% FBS. Then Hela cells were incubated with 100 μL of nanoparticles at different concentration after density of cells reached 80% in 96-well plates. The treatment groups include: GNRs@PDA-PEG group (10 μg/mL, 20 μg/mL and 30 μg/mL, based on gold ion concentration), GNRs@PDA-PEG + laser group (10 μg/mL, 20 μg/mL and 30 μg/mL, based on gold ion concentration), GNRs@PDA-PEG-DOX group (10 μg/mL, 20 μg/mL and 30 μg/mL, based on gold ion concentration), GNRs@PDA-PEG-DOX + laser group (10 μg/mL, 20 μg/mL and 30 μg/mL, based on gold ion concentration), laser group and PBS group. The power density of 808 nm laser is 2 W/cm2 and the irradiation time is 5 min. The cells were cultured in 37 °C and 5% CO2. Finally, the medium was removed and the HeLa cells were washed twice by PBS. The MTT solution was added into each well, forming the blue formazan crystals. After 2 hours, with the addition of DMSO, the blue



formazan crystals were dissolved. The absorption value was recorded by the microplate reader at a wavelength of 570 nm.

2.10

Calcein AM/PI staining. To further investigate the anti-tumor effect of GNRs@PDA-

PEG-DOX nanocomposites, the calcein AM/PI staining was employed. HeLa cells were seeded in 6-well plates (8 × 103 cells per well) and cultured in DMEM with 10% FBS under 37 °C with 5% CO2. Once the density of cells arrived at 80%, GNRs@PDA-PEG (100 μL, 20 μg/mL, based on gold ion) and GNRs@PDA-PEG-DOX (100 μL, 20 μg/mL, based on gold ion) were added to displace the medium. After incubation for 2 hours, the treatment groups were irradiated under an 808 nm laser with power density of 2 W/cm2 for 5 min. Afterwards, the medium was removed and the HeLa cells were washed three times by PBS. The double staining method was conducted for 20 min in 6-well plates. Lastly, the image of live and death was examined with an inverted fluorescence microscope.

2.11

Cellular uptake. 1 mL of HeLa cells were firstly seeded into 6-well plates and

incubated with DMEM (10%FBS) with the cell density of 1×104 cells/mL. After 12 hours, the GNRs@PDA-PEG-DOX nanocomposites (20 μg/mL, based on gold ion) were co-cultured with HeLa cell for 2 hours in the dark field. Then the cells were washed three times by PBS and costained by Hoechst 33342. After 15 min, the cells were washed by PBS and observed through a confocal microscope.


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2.12

Anti-migration in vitro. The HeLa cells were collected via centrifugation at 1000 rpm

for 5 min after digesting with trypsin. Then, the HeLa cells were seeded in 6-well plates with the density of 8 × 103 cells per well. After the cells arrived at 80% in 6-well plates under 37 °C with 5% CO2, 1 mL of the sterile pipette was utilized to scratch the monolayer cells. Afterwards, the cells were washed by PBS for twice and incubated with GNRs@PDA-PEG-DOX (1 mL, 5 μg/mL, based on gold ion), GNRs@PDA-PEG-DOX (1 mL, 5 μg/mL, based on gold ion) + laser, while the negative control group was treated with PBS. Finally, the wound of HeLa cells was examined with an inverted optical microscope.

2.13

The xenograft tumor models. The healthy female nude mice (about 20 g, 5-6 weeks

old) were purchased and fed under pathogen-free condition. The HeLa cells were obtained through centrifugation after split using 0.25% trypsin-EDTA. The 100 μL of cells with a density of 5×106 cells per mice were injected subcutaneously into the forelimb of the mice. The length and width of the tumor were measured daily to quantify the tumor growth. When the average tumor volume grew to 100 mm3, the xenograft-bearing mice were divided randomly into different groups.

2.14

Photoacoustic imaging. To investigate the targeting of nanoparticles to tumors,

photoacoustic imaging (PAI) was carried out by photoacoustic computed tomography scanner. The xenograft tumor mice had received 200 μL of GNRs@PDA-PEG-DOX (6 mg/kg) via intravenous injection and PBS as a negative control. The laser pulses with a wavelength of 800



nm were performed at 8 hours after injection, allowing maximum optical absorption of GNRs was utilized for PAI. Image reconstruction was conducted by OsiriX MD software.

2.15

In vivo photothermal evaluation. To examine the photothermal efficacy of

nanocomposites in vivo, the xenograft tumor mice were injected intravenously with PBS (200 μL), GNRs@PDA-PEG (200 μL, 6 mg/kg) and GNRs@PDA-PEG-DOX (200 μL, 6 mg/kg). 8 hours post injection, whole mice were exposed by 808 nm diode laser for 5 min (power density is 2 W/cm2). The thermal images of tumor site were captured in real-time by IR camera at a fixed time interval. Lastly, the Fotric AnalyzIR Software was utilized to analyze temperature changes of these thermographs.

2.16

In vivo antitumor study. To evaluate and investigate the photothermal tumor ablation

in vivo, the xenograft-bearing mice were assigned randomly into 4 groups: Control group (injection with PBS), Laser only group, GNRs@PDA-PEG-DOX group (injection with GNRs@PDA-PEG-DOX, 6 mg/kg, based on gold ion) and GNRs@PDA-PEG-DOX + laser group (injection with GNRs@PDA-PEG-DOX, 6 mg/kg, based on gold ion). Herein, the PTT treatment groups were subjected with NIR laser irradiation (the power density of laser is 2 W/cm2) for 5 min at 8 hours post-injection. Afterwards, the length and width of the tumor in mice were observed and recorded every time. The tumor volumes of mice were calculated from the equations: tumor volume = length × (width)2 × 1/2. The experiment procedure of tumorbearing mice was employed under protocols of the Institutional Animal Care and Use


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Committee (IACUC) of Nanjing University.

2.17

Examination of liver metastasis: To investigate suppression efficiency of

nanocarriers for hepatic metastasis, the xenograft-bearing mice were received treatment and sacrificed after long-term therapy. The livers were then collected and soaked in 4% (w/v) paraformaldehyde solution. The representative images in each group were photographed and the tumor metastasis sites were counted.

2.18

Histological examinations. For histology examination, the tissues and tumors of mice

were harvested immediately after the mice were sacrificed. After that, 4% (w/v) paraformaldehyde solution was used to fix for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining. All the sections were examined by an inverted light microscope.

2.19

Statistical evaluation. The data were analyzed by a one-way ANOVA followed by a

multiple comparison Bonferroni’s tests and shown as the mean ± standard deviation (SD). The value of *p

Gold Nanorods-Based Smart Nanoplatforms for Synergic

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