Acid-Triggered in Situ Aggregation of Gold Nanoparticles for

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Acid-Triggered in Situ Aggregation of Gold Nanoparticles for Multimodal Tumor Imaging and Photothermal Therapy Yumin Zhang, Jinglin Chang, Fan Huang, Li-Jun Yang, Chunhua Ren, Lin Ma, Wenxue Zhang, Hui Dong, Jinjian Liu, and Jianfeng Liu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01623 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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ACS Biomaterials Science & Engineering

Acid-Triggered in Situ Aggregation of Gold Nanoparticles for Multimodal Tumor Imaging and Photothermal Therapy

Yumin Zhanga#, Jinglin Changa#, Fan Huanga, Lijun Yanga, Chunhua Rena, Lin Maa, Wenxue Zhangb, Hui Donga*, Jinjian Liua*, Jianfeng Liu.a* #These

authors contributed equally to this work.

aTianjin

Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine,

Institute of Radiation Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College. Baidi Road 238, Nankai District, Tianjin 300192, PR China. bTianjin

Medical University General Hospital Radiation Oncology Department.

Anshan Road 154, Heping District, Tianjin 300052, PR China.

Corresponding Authors Jianfeng Liu, Tel: (+86)-022-85683019; E-mail: [email protected] Hui Dong, Tel: (+86)-022-85682389; E-mail: [email protected] Jianjian Liu, Tel: (+86)-022-85682399; E-mail: [email protected]

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ACS Biomaterials Science & Engineering 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

ABSTRACT Photothermal agents with high photothermal transfer efficiencies in the near-infrared (NIR) region are important for enhanced photothermal therapy (PTT) of tumors. Herein, we developed a strategy for the acid-triggered in situ aggregation of a system based on peptide-conjugated gold nanoparticles (GNPs). In an acidic environment, the GNPs formed large aggregates in solution, in cell lysates and in tumor tissues, as observed by transmission electron microscopy (TEM). As a consequence of the aggregation, their UV-Vis absorbance in the NIR region was greatly increased, and laser irradiation of the GNPs resulted in a dramatic increase in the temperatures of solutions and tumors that contained the GNPs system. When exposed to NIR irradiation, the aggregates formed by the GNPs system under acidic conditions were capable of producing a sufficient level of hyperthermia to destroy cancer cells both in vitro and in vivo. Interestingly, the GNPs aggregates showed enhanced properties in multiple imaging modalities, including computed tomography (CT), photoacoustic (PA) and photothermal (PT) imaging. Thus, we have developed a novel probe for enhanced multimodal tumor imaging. These findings prove that a strategy involving the acid-triggered in situ aggregation of a GNPs system can increase the photothermal transfer efficiency for low to high energy conversion, thus boosting the therapeutic specificity and antitumor efficacy of PTT and facilitating multimodal imaging.

KEYWORDS: Gold nanoparticles, Acid-triggered aggregation, Photothermal


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transfer efficiency, Multimodal imaging, Photothermal therapy

1. INTRODUCTION Among the promising recent antitumor strategies, photothermal therapy (PTT) has attracted much attention owing to its noninvasiveness, short treatment time, and specific tumor targeting ability.1-8 Local heating within the tumor site using near-infrared (NIR) irradiation and a photothermal agent can directly kill tumors with little harm to healthy tissues, thus providing a simple yet highly efficient antitumor strategy. To further improve the antitumor efficacy, it is necessary to develop photothermal agents with high photothermal transfer efficiencies in the NIR region.9-14 Several photothermal agents,15-19 such as cyanine dyes or fluorescent dyes with spectral absorptions in the NIR region, have been widely studied as photoacoustic (PA) contrast agents, photosensitizers and photothermal agents for PA imaging,20-23 photodynamic therapy (PDT),24-28 and photothermal therapy (PTT), respectively.29-31 However, various energy-level transition pathways are activated in fluorescent dyes under NIR irradiation, and their photothermal transfer efficiencies can therefore be greatly diminished under NIR excitation, resulting in the suboptimal phototherapy of tumors.18 The blood clearance rates and tumor accumulation of fluorescent dyes have been improved by assembling them into nanocarriers or by loading them onto nanoparticles.32-34 However, it is still necessary to solve the problem of controlling and optimizing the energy-level transition pathways that occur during the course of PDT or PTT.



Au-based nanomaterials are highly biocompatible and photostable.35, 36 They exhibit surface plasmon resonance, which improves the effectiveness of their optical-thermal conversion, and they are available in a variety of morphologies with size-dependent optical properties for tuning their absorption response in the near-infrared region. These features improve the flexibility and extend the applications of Au nanomaterials in PTT. Various Au-based nanomaterials have been prepared as photothermal agents to improve photothermal transfer efficiencies. In particular, compared to cyanine dyes, Au-based nanomaterials have fewer energy-level transition pathways under NIR irradiation,37 which enhances the localized thermal destruction of tumors owing to the high photothermal transfer specificity of Au-based nanomaterials. However, during the process of preparing Au-based nanomaterials such as gold nanorods,38, 39 gold nanostars 40, 41 and gold nanoprisms,42 it is necessary to use a series of highly toxic surfactants such as cetyltrimethylammonium bromide (CTAB).43, 44 These agents can damage healthy tissues due to their cytotoxicity and systemic toxicity, and thus, the clinical application of such nanostructures is limited. Furthermore, the therapeutic specificity of PTT depends on the specific delivery of NIR laser irradiation to tumors, and it remains impossible to differentiate the boundary between the tumor tissue and the surrounding normal tissues during the early treatment or postoperative treatment stages. In experiments with gold nanorods or gold nanostars, healthy surrounding tissues have been damaged by PTT as the nanoparticles arrive at the tumor site or as they diffuse out of the tumor.45, 46


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Recently, various stimulus-responsive nanocarriers or prodrugs have been successfully developed to achieve controlled drug delivery and stimulus-responsive drug release.47-50 These systems use the unique characteristics of the tumor microenvironment, such as high GSH, acidic pH, and overexpression of specific enzymes, to achieve controlled drug release in the tumors.51-53 As described in our previous study, DOX was conjugated to PEG by the formation of a Schiff base to prepare pH-sensitive antitumor prodrug nanoparticles (PEG-DOX NPs).54 These nanoparticles disassembled in the mildly acidic microenvironment of the cancer cells, thus simultaneously releasing DOX and the loaded cargo to achieve combination therapy.54 A novel strategy for the enzyme-induced aggregation of NPs as a drug carrier has been developed to achieve enhanced tumor retention and the sustained release of drugs.46

It is believed that the therapeutic specificity and efficiency of PTT can be improved by using these stimulus-responsive strategies.55-59 Therefore, we envisioned a strategy for the acid-triggered in situ aggregation of pH-responsive gold nanoparticles (GNPs). After arriving in a tumor, aggregates of the GNPs would be immediately formed in the acidic tumor environment. These GNPs aggregates would have a greatly increased absorptivity in the NIR region between 650 and 900 nm, thus achieving an enhanced photothermal transfer efficiency. In nontumor tissues, the GNPs would not assemble into aggregates, and they would retain their low photothermal transfer efficiency.



Therefore, the stimulus-responsive switch of the photothermal transfer efficiency from low to high would reduce the damage to off-target tissues.

Herein, we describe the preparation of a GNPs system, based on the acid-triggered aggregation of ~30 nm GNPs, to serve as a novel photothermal agent for enhanced tumor PTT. The system contains GNPs-A, which are modified with an Asp-Asp-Asp-Asp-Asp-Cys peptide, and GNPs-B, which are modified with a Lys-Lys-Lys-Lys-Lys-Cys peptide grafted to 2,3-dimethylmaleic anhydride (DA). In an acidic environment, the surface of GNPs-B was converted from a negatively charged state to a positively charged state, and GNPs-B then electrostatically interacted with the negatively charged GNPs-A to form large GNPs aggregates. Notably, as the GNPs aggregates assembled, the absorption in the NIR region was greatly enhanced compared to that of individualized GNPs. Thus, the photothermal transfer efficiency of the GNPs under NIR excitation was improved, leading to enhanced antitumor specificity and efficacy during PTT. In addition, the computed tomographic (CT) imaging and photoacoustic (PA) imaging signals of the GNPs were improved as the aggregate size increased, thus facilitating improved multimodal imaging. Therefore, this GNPs system, in which the photothermal transfer efficiency is controlled by the acid-triggered aggregation of the GNPs in tumors, demonstrates great potential for use as a multimodal imaging agent and in enhanced PTT.


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Scheme 1. Diagram depicting the composition of the GNPs system and the acid-triggered in situ aggregation of the GNPs. The mechanism of charge reversal for the surface of GNPs-B (A). GNPs-B undergoes charge reversal when exposed to an acidic environment and then electrostatically interacts with GNPs-A to form aggregates. A schematic illustration of the enhanced multimodal tumor imaging and photothermal therapy due to the increased absorption of the GNPs in the NIR region (B).

2. EXPERIMENTAL SECTION 2.1 Materials. HAuCl4·3H2O was provided by Sigma Aldrich (USA). The ACS Paragon Plus Environment


Asp-Asp-Asp-Asp-Asp-Cys peptide and Lys-Lys-Lys-Lys-Lys-Cys peptide were purchased from GL Biochem Ltd. (Shanghai, China). 2,3-Dimethylmaleic anhydride (DA) was purchased from J&K Scientific Ltd. (Beijing, China), and mPEG-SH with a molecular weight of 2000 kDa was obtained from Yare Biotech (Shanghai, China). All other chemical reagents were of analytical grade and were used without further purification.

2.2 Preparation and Characterization of the GNPs System. First, 100 mg of peptide B (Lys-Lys-Lys-Lys-Lys-Cys) and 206 mg of 2,3-dimethylmaleic anhydride (DA) were dissolved in PBS (pH 8.0) and slowly stirred for 24 h at room temperature to synthesize the DA-grafted peptide B. The excess DA was removed by dialysis. The white powder of the DA-grafted peptide B was then obtained by an ultralow-temperature freeze-drying method, and its structure was characterized by using 1H NMR (Varian INOVA). Then, peptide A (Asp-Asp-Asp-Asp-Asp-Cys, 1.75 mg) and DA-grafted peptide B (2.5 mg) were conjugated separately onto the GNPs (180 μg/mL, 10 mL) at pH 8.0 with stirring for 24 h at room temperature. GNPs-A and GNPs-B were both purified by centrifugation at 6000 rpm (10 min) for three times, and the collected supernatants were measured by the BCA method to indirectly detect the peptide concentrations of GNPs-A and GNPs-B. The GNPs system was finally obtained by mixing GNPs-A and GNPs-B with the same concentration of GNPs. Additionally, GNPs conjugated to PEG2000 (GNPs-PEG2000) were synthesized by the same method. The size distributions and zeta potentials of the


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prepared GNPs-system and the GNPs-PEG2000 were measured by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS).

To verify the successful preparation of the GNPs system, the GNPs were first characterized using a Varioskan Flash microplate reader (Thermo Scientific), UV-Vis spectrometry (Purkinje General), DLS (Malvern Zetasizer Nano ZS) and TEM (JEM-2100F). Afterwards, to analyze the acid-triggered aggregation of the GNPs, changes in various parameters of the GNPs system were measured at pH 6.5, including their UV-Vis spectrum, size and morphology. GNPs-PEG2000 was used as a control.

2.3 Photothermal Heating Experiments. Before investigating the photothermal heating efficiency of the GNPs system, its UV-Vis spectra in the pH range of 7.4 to 6.5 were first studied using a Varioskan Flash microplate reader (Thermo Scientific). Afterwards, different concentrations of the GNPs system (0, 90, 180, and 360 μg/mL) at pH 7.4 and pH 6.5 were irradiated with a continuous-wave NIR diode laser (808 nm, Qingdao Boguang Electronics Technology, China) at a power density of 2.5 W/cm² for 10 min, and GNPs-PEG2000 was used as the control. Then, the GNPs system (180 μg/mL) at pH 6.5 was irradiated at a series of power densities (0.5, 1, 2, and 2.5 W/cm²) for 10 min. Lastly, the GNPs system was subjected to five cycles of laser irradiation at a power density of 2.5 W/cm² for 10 min each cycle. The temperatures of the solutions were measured every 30 seconds using an infrared



thermal camera (Fluke Ti 200, Fluke Corp, Washington, USA).

2.4 Cellular Aggregation of the GNPs System. MCF-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) plus 10 % fetal bovine serum (FBS), streptomycin (0.1 mg/mL), and penicillin (100 U/mL) at 37 °C in a humidified incubator with 5 % CO2.

MCF-7 cells were seeded into 6-well plates at approximately 2×105 cells per well and incubated for 24 h. Then, the culture medium was replaced by various GNPs formulations (GNPs-PEG2000 or the GNPs system) in DMEM (180 μg/mL), and the cells were incubated for an additional 12 h. Afterwards, the samples were divided into four groups to investigate the cellular aggregation of the GNPs system in different experiments. First, the cells were washed with PBS, trypsinized, centrifuged, and fixed with 2.5 % glutaraldehyde. After 2 h of fixation at 4 °C, the samples were washed three times with PBS (0.02 M, pH 7.4). Then, the samples were fixed with 1 % perosmic oxide for 2 h at 4 °C. After being washed in water, the samples were dehydrated in a series of alcohols, embedded, and sliced to a thickness of 50 to 70 nm. The morphology of the GNPs system within the MCF-7 cells was imaged using TEM (Hitachi H-600, Japan) at 200 kV. Second, the cells were washed in a cold phosphate buffered saline (PBS) solution and then collected in EP tubes. The cells were lysed and treated with aqua regia (HCl:HNO3 = 1:3, volume ratio) for 2 h, and the Au concentration of each well was measured using inductively coupled plasma-atomic


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emission spectrometry (ICP-AES, ThermoFisher iCAP7400). Third, the UV-Vis spectra of cell lysates containing either the GNPs system or GNPs-PEG2000 were obtained with a Varioskan Flash microplate reader to investigate the variations in their absorbances in the NIR region. Finally, in the GNPs system group, cells were lysed in a cell lysis buffer for 0.5 h at 4 °C. After filtration with a filter membrane of 0.22 μm, the size distribution of the cell lysate was measured by DLS, for which the GNPs system and blank cell lysate were used as controls.

2.5 In Vitro Antitumor Effect of GNPs System. For the in vitro PTT study, MCF-7 cells were seeded into confocal culture dishes at a density of 105 cells per dish and cultured for 24 h at 37 °C in a humidified incubator with 5 % CO2. Then, the cells were incubated with the GNPs system and GNPs-PEG2000 (200 µg/mL) for 3 h and exposed to laser irradiation (808 nm, 2.5 W/cm², 6 min). PI (propidium iodide) and calcein AM (calcein acetoxymethyl ester) were used to stain the dead and living cells, respectively, which were then imaged by laser scanning confocal microscopy (Zeiss, LSM710).

For the cell viability assay, MCF-7 cells were cultured in RPMI 1640 medium supplemented with 10 % FBS at 37 °C in 5 % CO2. The cells were cultured in 96-well plates for 24 h and then incubated with the GNPs system at different concentrations (0, 5, 10, 20, 50, 100 and 200 µg/mL) for another 3 h. GNPs-PEG2000 was used as the control. Cells were laser-irradiated at 808 nm (2.5 W/cm²) for 6 min with a spot size



of 15 mm and then cultured for another 1 h. The methyl thiazolyl tetrazolium (MTT) reagent was added according to the instructions from the manufacturer and incubated for 4 h. The absorbance of the mixture at 490 nm was determined by a Varioskan Flash microplate reader.

2.6 In Vivo Multimodal Imaging of the GNPs System. Female BALB/c nude mice aged 6 weeks (Vital River Laboratories, Beijing) were subcutaneously injected with a suspension of MCF-7 tumor cells (2×106) in PBS. The tumors were allowed to grow until they reached a suitable size for multimodal imaging (150-200 mm3), and the mice were then divided into 2 groups (n = 3 per group). The GNPs system and GNPs-PEG2000 were intratumorally injected at a GNPs concentration of 1.0 mg/mL (100 μL). At 0.5 h postinjection, the aggregation behaviors of the GNPs system and GNPs-PEG2000 within the tumor were analyzed by TEM. In vivo multimodal imaging was carried out as follows. For computerized tomography (CT) imaging, the GNPs system and GNPs-PEG2000 (100 μL, 10 mg/mL) were intratumorally injected. CT scans and image analysis were conducted using a CT scanning system (Nanoscan, SPECT/CT) at 0.5 h postinjection. Photoacoustic (PA) imaging was performed using a multispectral optical tomography system (MSOT inVision 128, iThera Medical, Germany) at 5 min, 10 min, 0.5 h, 1 h, 1.5 h and 2 h postinjection of the GNPs system and GNPs-PEG2000 at a GNPs concentration of 1.0 mg/mL (100 μL). For photothermal (PT) imaging, the tumors in the mice were treated with NIR laser irradiation (808 nm, 2.5 W/cm2) using a laser spot size of 8 mm to irradiate the whole


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tumor. The tumor temperatures were recorded using a Fluke Ti200 thermal imager. During these experiments, the mice were anesthetized with an intraperitoneal injection of 4 % chloral hydrate at a dose of 165 μL/20 g body weight. All animal studies were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (Tianjin, revised in June 2004).

2.7 In Vivo Photothermal Therapy. A MCF-7 xenograft mouse model was established as described above. When the tumor sizes reached approximately 100 mm3, the breast tumor-bearing mice were randomly divided into 6 groups (n=6): PBS with and without laser irradiation, GNPs-PEG2000 with and without laser irradiation, and GNPs system with and without laser irradiation. The mice were then intratumorally injected with 100 µL of the PBS, GNPs-PEG2000 or GNPs system (1.0 mg/mL). For the laser irradiation groups, the tumors were irradiated with an 808 nm laser at a power density of 2.5 W/cm² (5 min) at 0.5 h postinjection. The laser spot size used for therapy was 8 mm to irradiate the entire tumor. The tumor sizes were measured with a digital caliper every other day and their volumes were calculated using the formula: volume = (tumor length) × (tumor width)²/2. Relative tumor volume was calculated based on v/v0 (where v0 is the initial tumor volume). Hematoxylin and eosin (H&E) staining was used to assess the tissue lesions after PTT. The tumors and major organs (heart, liver, spleen, lungs and kidney) of the mice were collected, fixed in 4 % neutral formaldehyde, embedded in paraffin, cut into sections, stained with H&E and then observed under a microscope.



2.8 Statistical Analysis. SPSS 16.0 software was applied for the statistical analysis. All data are presented as the mean ± standard deviation values. The statistical significance between values was determined by the Student’s t test, for which *p

Acid-Triggered in Situ Aggregation of Gold Nanoparticles for

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