A Multifunctional Platform for Tumor Angiogenesis-Targeted Chemo

Nov 7, 2016 - Image-guided combined chemo-thermal therapy assists in optimizing treatment time, enhancing therapeutic efficiency, and circumventing si...
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A Multifunctional Platform for Tumor Angiogenesis-Targeted Chemo-Thermal Therapy Using Polydopamine-Coated Gold Nanorods Lu Zhang,† Huilan Su,‡ Jiali Cai,§ Dengfeng Cheng,⊥ Yongjie Ma,∥ Jianping Zhang,∇ Chuanqing Zhou,∥ Shiyuan Liu,§ Hongcheng Shi,⊥ Yingjian Zhang,∇ and Chunfu Zhang*,†,# †

State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China ‡ State Key Laboratory of Metal Matrix Composites and ∥School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China § Changzheng Hospital, Secondary Military Medical University, Shanghai 200003, China ⊥ Department of Nuclear Medicine, Zhongshan Hospital, Shanghai Medical College and ∇Department of Nuclear Medicine, Shanghai Cancer Center, Fudan University, Shanghai200032, China # Department of Nuclear Medicine, Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China S Supporting Information *

ABSTRACT: Image-guided combined chemo-thermal therapy assists in optimizing treatment time, enhancing therapeutic efficiency, and circumventing side effects. In the present study, we developed a chemo-photothermal theranostic platform based on polydopamine (PDA)-coated gold nanorods (GNRs). The PDA coating was thin; however, it significantly suppressed the cytotoxicity of the cetyltrimethylammonium bromide template and allowed high cisplatin loading efficiency, arginine-glycine-aspartic acid (RGD) peptide (c(RGDyC)) conjugation, and chelator-free iodine-125 labeling (RGD-125IPt-PDA@GNRs). While loaded cisplatin was released in a pH-sensitive manner, labeled 125I was outstandingly stable under biological conditions. RGD-125IPt-PDA@GNRs had a high specificity for αvβ3 integrin, and consequently, they could selectively accumulate in tumors, as revealed by single photon emission computed tomography/CT imaging, and in target tumor angiogenic vessels, as shown by high-resolution photoacoustic imaging. As RGD-125IPt-PDA@GNRs targets tumor angiogenesis, it is a highly potent tumor therapy. Combined chemo-photothermal therapy with probes could thoroughly ablate tumors and inhibit tumor relapse via a synergistic antitumor effect. Our studies demonstrated that RGD-125IPt-PDA@GNRs is a robust platform for image-guided, chemo-thermal tumor therapy with outstanding synergistic tumor killing and relapse inhibition effects. KEYWORDS: gold nanorods, cisplatin, polydopamine, image-guided, angiogenesis targeted, photoacoustic imaging, chemo-photothermal combined therapy various structures, such as gold nanovesicles,2 gold nanoshells,3 gold nanorods (GNRs)4 and gold nanocages,5 have received much attention in the recent years. Among them, GNRs have attracted more attention because of advantageous features like high optical absorption coefficients in the NIR region, precisely tunable light absorption range via the adjustment of their aspect

P

hotothermal therapy (PTT) of tumors is a potentially curative treatment modality that has been undergoing rapid technological advancements owing to the advent of image-guided procedures and nanotechnology. This modality feasibly involves minimal invasion using exogenous optical absorber designs. As the major tissue chromophores are minimally absorptive in the near-infrared (NIR) range (650− 900 nm),1 nanomaterials with absorptions in this spectral region and good photothermal conversion efficiencies are often designed for PTT. In this regard, gold nanomaterials with © 2016 American Chemical Society

Received: September 16, 2016 Accepted: November 7, 2016 Published: November 7, 2016 10404

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ACS Nano ratios, and efficient large-scale synthesis.6 Particularly, GNRs have been demonstrated to be one of the most efficient exogenous agents for NIR photothermal therapy of cancers, showing high efficacy in the ablation of cancer cells in vitro or tumors in vivo.7−9 However, PTT has some notable limitations. Because of the distribution of energy only within the beam, the heat produced by NIR irradiation from the GNRs reduces gradually in the tissues. This therapy also shows less-than-ideal soft tissue penetration caused by the inevitable depth-dependent decline of laser intensity and soft tissue absorption. These weaknesses lead to insufficient tumor cell killing and failure to prevent tumor recurrence by initial treatment.10 In order to increase the therapeutic efficacy, GNR-based synergistic or combined therapies,11,12 such as chemo-photothermal therapy, have been developed. To this end, various materials have been explored for coating GNRs and loading anticancer drugs. Although combined therapy has been shown to improve therapeutic efficacy better than single therapy alone, coating dramatically increases the particle size, which reduces the tumor accumulation efficiency of the particles through the enhanced permeability and retention (EPR) effect.13,14 In addition, to experimentally determine the optimal time point with maximal particle accumulation within the tumor, GNRs are often labeled with gadolinium or radioisotopes (e.g., 99m Tc, 125I, and 64 Cu), rendering them detectable by magnetic resonance imaging (MRI) or nuclear imaging (single photon emission computed tomography [SPECT]/ positron emission tomography [PET]). However, the particles are conventionally labeled with both gadolinium and radioisotopes through a chelator-mediated procedure, which would further increase the size of GNRs and complicate probe preparation. To facilitate labeling, a simple but effective chelator-free strategy is highly desirable.15−17 For example, it has been reported that gold nanoparticles (GNPs) and GNRs could be directly labeled with iodine-125 via physical absorption.18 However, the labeling was not stable, and iodine-125 easily detached from the particles under biological conditions. Therefore, a versatile coating layer that allows efficient and highly stable drug loading and chelatorfree radioisotope labeling, but does not markedly increase the particle size, is highly desirable for GNR-based chemo-thermal therapy. Since Messersmith et al. reported the mussel-inspired adhesive protein for multifunctional coating in 2007,19 polydopamine (PDA) has received extensive attention owing to its extremely attractive properties of self-polymerization and spontaneous deposition on the surface of virtually any material under alkaline conditions to form a designable and flexible layer.20,21 Moreover, PDA exhibits versatile chemical reactivity during secondary reactions.20 For instance, it can bind metal ions and form a metal coating owing to the binding ability of catechols in PDA. Moreover, quinone groups in PDA can also react with thiols or amines via the Michael addition or Schiff bases reaction,19 which facilitates the modification of particles with antibiofouling polymers such as polyethylene glycol (PEG) and further functionalizes them with targeting molecules.22,23 Therefore, in the present study, we coated GNRs with PDA, explored the potential of PDA coating layer for PEG surface modification, cisplatin loading, and chelator-free iodine-125 labeling, and developed a tumor angiogenesis-targeted theranostic platform based on the PDA-coated GNRs. This platform demonstrates three distinct features: (1) high cisplatin

loading efficiency with pH-sensitive release, resulting in selective drug delivery to the tumor environment; (2) high chelator-free iodine-125 labeling stability, permitting SPECT/ CT-guided chemo-photothermal therapy of cancers; and (3) high specificity in targeting tumor angiogenesis, leading to sufficient ablation of tumors via chemo-photothermal combined therapy.

RESULTS AND DISCUSSION Preparation and Characterization of RGD-125IPt-PDA@ GNRs. GNRs were synthesized via a seed-mediated growth procedure.6 Initially, gold nanoseeds measuring approximately 5 nm in size were synthesized, upon which GNRs were grown via a cetyltrimethylammonium bromide (CTAB) template. The GNRs were uniform in size with an average length of 54 ± 2 nm and diameter of 15 ± 1 nm (aspect ratio of ∼3.5; Figure 1A). The transverse and longitudinal local surface plasmon

Figure 1. Characterization of GNRs. (A, C, and D) TEM images of GNRs before (A) and after (C, D) PDA coating. (B) UV−vis spectra of GNRs, Pt-PDA@GNRs, and RGD-IPt-PDA@GNRs.

resonance (LSPR) peaks were approximately 520 and 810 nm, respectively (Figure 1B). Owing to the surface CTAB coating, the zeta potential of the GNRs was 29.8 mV (Table S1). In order to suppress the cytotoxicity of CTAB and enable further surface engineering,24 we coated the GNRs with PDA (Figure 1C, D). The PDA-coated layer was uniform and approximately 1−2 nm in thickness (Figure 1D). The zeta potential of the PDA-coated GNRs (PDA@GNRs) was 24.1 mV (Table S1). The LSPR peak of the PDA@GNRs was estimated by UV−vis spectroscopy (Figure 1B), which indicated that no significant shift occurred after the coating. The PDA coating was achieved by self-polymerization of dopamine under alkaline conditions.25,26 Under the alkaline conditions (pH > 7.5), dopamine is first oxidized to the highly reactive dopamine quinone. The pendant amine of dopamine quinone is condensed to form 5,6-dihydroxyindoline that further undergoes oxidization and rearrangement to form 5,6dihydroxyindole, which is easily oxidized to 5,6-indolequinone. The 5,6-dihydroxyindole and 5,6-indolequinone are capable of forming oligomers between catechol and o-quinone via the reverse dismutation reaction, which results in a layer of PDA coating. However, the molecular mechanisms behind the formation of PDA and its structure are complex and remain 10405

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Figure 2. A characterization of the RGD-IPt-PDA@GNRs probes. (A) Quantitative analysis of cisplatin loading on PDA@GNRs by atomic adsorption spectroscopy. (B) The release profiles of cisplatin from the probes in PBS at different pH values. (C) TEM images of RGD-IPtPDA@GNRs. (D, E) Element mappings of Au (D) and Pt (E) of RGD-IPt-PDA@GNRs. (F) Radioactive stability of iodine-125 on the probes in mouse serum. (G) TEM images of RGD-IPt-PDA@GNRs after mixing into the cell culture medium (pH = 7.4) for 15 days. Insert: Highresolution TEM image of the probe.

controversial.25,26 Many functional groups including catechol or quinone functions, amino groups, planar indole units, and carboxylic acid groups might integrate to form PDA, which provides a versatile platform for further loading of conjugates with other interesting functionalities.21 To improve the in vivo behavior of PDA@GNRs and provide a means for surface functionalization, the PDA@GNRs were modified with amino-poly(ethylene glycol)-thiol (NH2-PEGSH; MW 5000). Herein, the sulfhydryl group reacted with the quinone in PDA via the Michael addition reaction, forming an irreversible neighboring covalent bond in the absence of harsh reaction conditions,27 thereby anchoring the NH2-PEG-SH moiety on the surface of the PDA@GNRs to the amino group readily exposed in the solution. After PEG coating, the zeta potential of the PDA@GNRs diminished from 24.1 mV to −9.43 mV (Table S1). The catechol groups in PDA have been demonstrated to be able to chelate metal ions28 and exhibit a pH-responsive release property.29 Cisplatin is one of the most commonly used chemotherapy drugs in the treatment of solid tumors30 and hydrolyzes under aqueous conditions, forming a platinumammonia hydrated ion ([Pt(H2O)2(NH2)2]2+).31 Following PEG modification, we explored the potential of PDA@GNRs for cisplatin loading. For this purpose, the PEGylated PDA@ GNRs (1 mg) were suspended in an aqueous solution of cisplatin (0.3 mL) with different concentrations and stirred for

12 h at room temperature. As shown in Figure 2A, the loading amount of cisplatin increased with increasing cisplatin concentration. At the concentrations of 0.6 and 1 mg/mL, 89 ± 1 μg and 104 ± 3 μg cisplatin was loaded, respectively, which are much higher than that loaded on mercaptoundecanoic acid (MUA)-capped GNPs32 or by ligand exchange method.33 Only a marginal increase in Pt loading was achieved by further increasing the concentration of cisplatin to 1.5 mg/mL. Therefore, in the subsequent experiments, we loaded the PEGylated-PDA@GNRs with 1 mg/mL cisplatin (Pt-PDA@ GNRs). An energy-dispersive X-ray spectroscopy (EDS) element mapping of the loaded GNRs was performed to confirm the presence of Pt element on the surface of PDA@GNRs (Figure 2C−E). As shown in Figure 2D, E, the red dots indicative of Au element and green dots indicative of Pt element were observed in the same region. These element dots could overlap with the transmission electron microscope (TEM) image of the GNRs (Figure 2C), demonstrating the distribution of Pt element on PDA@GNRs. Cisplatin loading may be achieved by coordination of [Pt(H2O)2(NH2)2]2+ with 5,6-dihydroxyl groups in catechol on the layer of PDA coating, thus forming catechol−Pt complexes.20,21 To conjugate the arginine-glycine-aspartic acid (RGD) peptides (c(RGDyC)) to the GNRs, the PEGylated PtPDA@GNRs were first activated with sulfosuccinimidyl-4-[N10406

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ACS Nano Scheme 1. Schematic Illustration of the RGD-125IPt-PDA@GNRs Probe Preparation Procedure

using the same procedure for iodine-125 labeling, to serve as cold probes. Arginine-alanine-aspartic acid (RAD) peptide (c(RADyC))-coupled, 125I- or I-labeled Pt-PDA@GNRs were also prepared as control probes (RAD-125IPt-PDA@GNRs, RAD-IPt-PDA@GNRs). The LSPR peaks of the final product did not shift (Figure 1B). The integrity of the PDA coating on the GNRs was also investigated under biological conditions. To this end, RGD-IPtPDA@GNRs were dispersed in DMEM (with 10% FBS) cell culture media under normal (pH = 7.4) or acidic (pH = 5.4) conditions, stirring for 15 days, and then examined via TEM. As shown in Figures 2G and S2, the PDA coating remained intact, and no obvious detachment was observed under either condition. These observations are consistent with the results of previous studies, which reported that the PDA coating could serve as an ultrastable modification on the surface of nanomaterials, thus improving their biocompatibility.37 To study the release behavior of the loaded cisplatin, the RGD-IPt-PDA@GNRs were suspended in PBS at different pH levels at 37 °C (Figure 2B). The loaded cisplatin was nearly unreleased at pH 8.5. However, under more acidic conditions (pH = 7.4 and 6), the cisplatin showed a favorable sustained release from the GNRs. Twenty hours post-incubation, 24.4 ± 0.3% and 51.3 ± 2.4% of the loaded cisplatin was released at pH 7.4 and pH 6, respectively. Under acidic conditions, hydrogen ions [H+] would interact with the lone electron pairs of the oxygen ions in the hydroxyl groups of catechol, leading to the detachment of [Pt(H2O)2(NH2)2]2+. The pH-sensitive release of cisplatin was consistent with previous reports28,38 and confirmed our speculation that cisplatin was loaded onto PDA@GNRs through the coordination of [Pt-

maleimidomethyl]-cyclohexane-1-carboxylate (sulfo-SMCC), and the peptides were then covalently coupled to the GNRs through thiol-maleimide linkages.34 The zeta potential of the RGD peptide functionalized Pt-PDA@GNRs (RGD-Pt-PDA@ GNRs) was −31.3 mV (Table S1). The efficiencies of PEG modification and subsequent RGD peptide conjugation on the PDA@GNRs were estimated using the Elleman method.35 As shown in Figure S1, both the PEG modification and RGD peptide conjugation were highly efficient. We determined the numbers of PEG molecules and RGD peptides immobilized on a GNR to be 98 ± 23 and 58 ± 11, respectively. To label RGD-Pt-PDA@GNRs with radioisotope iodine-125, iodine-125 (Na125I, 300 μCi) was added into the RGD-PtPDA@GNRs suspension (0.3 mL, 4 mg/mL) and incubated for 30 min. 125I labeling was verified by radio-thin-layer chromatography, and the labeling efficiency was determined to be approximately 100%. The final product, designated as RGD-125IPt-PDA@GNRs, was purified using size exclusion filters and size exclusion chromatography with disposable columns containing Sephadex G-25 medium and saline as the eluent. The purified RGD-125IPt-PDA@GNRs were highly stable in fetal bovine serum (FBS), and almost no iodine-125 detachment was detected after incubation at 37 °C for 24 h (Figure 2F), which is significantly better than the conventional tyrosine-mediated labeling and physical absorption method.18,36 Iodine-125 was speculated to directly bind quinone or indolines via the formation of an irreversible neighboring covalent bond, thus resulting in stable complexes. The overall procedure for the preparation of RGD-125IPt-PDA@GNRs is outlined in Scheme 1. Meanwhile, the RGD-Pt-PDA@GNRs were also labeled with nonradioactive iodine (RGD-IPt-PDA@GNRs) 10407

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Figure 3. Specificity of the probes for αvβ3 integrin. (A) Two-photon microscope images of H1299 cells treated with RGD-IPt-PDA@GNRs, RAD-IPt-PDA@GNRs, and RGD-IPt-PDA@GNRs plus free RGD peptide (10 μM) at a concentration of 50 μg Au/mL for 1 h. Scale bar: 20 μm. (B) Orthogonal projections of the z-stack reconstructions showing relative position of GNRs (green) compared to the cell body (blue, DAPI staining for cell nuclei). Scale bar: 20 μm. (C) Intracellular Au contents quantified by atomic adsorption spectroscopy. **, p < 0.01.

Figure 4. Chemotherapeutic treatment of tumor cells. (A−C) Viability of H1299 cells after incubation with RGD-IPt-PDA@GNRs, RGD-IPDA@GNRs, and pristine GNRs at different concentrations (10, 25, 50, and 100 μg Au/mL) for 6 h (A) or at the concentration of 50 μg Au/ mL for different durations (B and C). **, p < 0.01.

(H2O)2(NH2)2]2+ with the oxygen ions of the hydroxyl groups of catechol in the PDA coating. Because the longitudinal LSPR peak of the GNRs was approximately 810 nm, we estimated the photothermal conversion performance of RGD-IPt-PDA@GNRs irradiated by an 808 nm laser in the test tubes. As expected, the RGD-IPtPDA@GNRs suspensions exhibited both laser energy and a GNR concentration-dependent photothermal conversion effect (Figure S3A, B). Compared to the pristine GNRs at the same concentration, RGD-IPt-PDA@GNRs exhibited no significant variation of photothermal conversion efficiency. To examine whether heating could damage the morphology of the GNRs, TEM examinations of RGD-IPt-PDA@GNRs were performed before and after irradiation at 7 W/cm2 at the concentration of 12.5 μg/mL for 10 min. Compared to the probes before irradiation (Figure S3C), no obvious morphological change was detected after irradiation, and the PDA coating maintained its integrity, although the temperature rose to approximately 90 °C (Figure S3D). Our results demonstrated that RGD-IPt-PDA@ GNRs have high photothermal conversion efficiencies and good

stability at high temperature, which render the probes very promising as photothermal therapeutic agents. Specificity of the Probes. To evaluate the specificity of the probes for the αvβ3 integrin, H1299 cells (a nonsmall-lungcancer cell line, αvβ3 integrin positive)39 were treated with RGD-IPt-PDA@GNRs, RAD-IPt-PDA@GNRs, or RGD-IPtPDA@GNRs plus free RGD peptide at a concentration of 50 μg Au/mL for 1 h. Subsequently, the cellular uptakes of these probes were evaluated using a two-photon microscope using sequential imaging along the z-axis at 0.45 μm interslice distances and quantified with an atomic adsorption spectrophotometer (AAS). Both two-photon microscopy (Figure 3A, representative cell images from the middle slice of the cells) and AAS quantification (Figure 3C) indicated that the cellular uptake of RGD-IPt-PDA@GNRs was greater than that of RADIPt-PDA@GNRs, and the uptake could be significantly inhibited by free RGD peptides. These results coherently indicated that RGD-IPt-PDA@GNRs could specifically target αvβ3 integrin positive cells, and the cellular uptake of the probes was mainly mediated by the αvβ3 integrin.40 Moreover, orthogonal projections of the z-stack reconstructions of cell 10408

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ACS Nano images revealed that RGD-IPt-PDA@GNRs were internalized by the cells rather than being associated with the cell surface (Figure 3B). Cytotoxicity and Chemotherapeutic Effect of the Probes in Vitro. The cytotoxicity and chemotherapeutic effect of the probes were evaluated using a cell counting kit-8 (CCK8) assay (Figure 4). For this purpose, H1299 cells were treated with RGD-IPt-PDA@GNRs at different concentrations for 6 h, or at a concentration of 50 μg Au/mL for different periods of time, using the pristine GNRs and RGD-I-PDA@GNRs as controls. The CTAB template on the surface of the pristine GNRs is hardly reduced by water-wash.41 Consistent with previous reports, the pristine GNRs demonstrated time- and dose-dependent cytotoxicity.42,43 Cell death was noticeable after treatment with the pristine GNRs at a concentration of 25 μg Au/mL for 6 h or 50 μg Au/mL for 1 h. The PDA coating significantly suppressed the cytotoxicity of the pristine GNRs, and cell death was marginal even after the cells were incubated with RGD-I-PDA@GNRs at a concentration of 50 μg Au/mL for 120 h. However, after cisplatin loading, RGD-IPt-PDA@ GNRs showed significant cytotoxicity 96 h post-incubation, with a cell viability of 77.36 ± 2.10% (p < 0.01), and an even greater level of cytotoxicity 120 h post-incubation, with a cell viability of 43.86 ± 2.45% (p < 0.01). These results indicated that the loaded cisplatin might have a chemotherapeutic effect on the cells. Cisplatin is a chemotherapy drug that can react with the N7 atom of guanine and form DNA adducts, resulting in double-strand breaks in the DNA, which leads to apoptosis.30,44 To verify these results, we examined the treated cells with immunohistochemistry using antibodies against γH2AX, which is a generally accepted marker for double-strand breaks in DNA45 induced by radiotherapy46,47 or chemotherapy.32 We found that both free cisplatin and RGD-IPtPDA@GNRs remarkably induced DNA damage 72 h posttreatment, with RGD-IPt-PDA@GNRs showing a higher potency (Figure 5), while no obvious DNA damage was detected 36 h post-treatment (Figure S4). As previously demonstrated, RGD-IPt-PDA@GNRs specifically targeted the H1299 cells and initiated cell uptake of the probes. The internalized probes released the loaded cisplatin into endosomes. The free or dissociated cisplatin became activated intracellularly via aquation and diffused into the nuclei through nuclear pores, where it subsequently covalently bound to DNA, resulting in DNA damage.31 Photothermal and Chemo-Photothermal Therapy Effects in Vitro. To investigate the photothermal therapeutic effect of the probes in vitro, H1299 cells were incubated with the cold probe RGD-IPt-PDA@GNRs at a concentration of 50 μg Au/mL for 2 h. Subsequently, the culture medium was removed, the cells were washed three times, replenished with fresh culture medium, and irradiated with an 808 nm laser (2 Wcm−2) at different time points (5, 10, and 15 min). The irradiated cells were stained using calcein AM and propidium iodide (PI) to detect the live or dead cells. The confocal laser scanning microscopy images of the stained cells indicated that cell death was unnoticeable after 5 min of irradiation treatment; however, most cells were dead after laser irradiation for 10 or 15 min (Figure 6A). The cell viabilities after 5, 10, and 15 min laser irradiation were 81.25 ± 3.46%, 39.57 ± 2.12%, and 28.47 ± 4.72%, respectively (Figure 6B). These observations indicated that RGD-IPt-PDA@GNRs could efficiently convert NIR light into heat and reduce the viability of tumor cells in vitro. To investigate whether RGD-IPt-PDA@GNRs have

Figure 5. γ-H2AX immunostaining of H1299 cells treated with pristine GNRs, free cisplatin (5.78 μg Pt/mL), and the probes (50 μg Au/mL containing 5.78 μg Pt/mL) for 72 h. Scale bar: 20 μm.

synergistic chemo-thermal therapeutic effects on cancer cells, the H1299 cells were treated with 50 μg Au/mL of RGD-IPtPDA@GNRs for 2 h using RGD-I-PDA@GNRs as a control. The treated cells were irradiated with a 808 nm laser light for 10 min and then maintained for different periods of time. Cell viabilities were evaluated using CCK-8 analysis. As shown in Figure 6C, 12 h post-laser irradiation, the viabilities of RGD-IPDA@GNRs- and RGD-IPt-PDA@GNRs-treated cells were 29.35 ± 0.81% and 36.42 ± 3.34%, respectively, indicating that the cells were killed mainly via the photothermal effect of GNRs. By extending the maintenance time, the viability of RGD-I-PDA@GNRs-treated cells gradually recovered from 29.35% (12 h) to 85.02% (72 h). In contrast, RGD-IPt-PDA@ GNRs-treated cells remained approximately 40% viable. These results indicated that in addition to its thermal effect, RGD-IPtPDA@GNRs could inhibit cell viability by cisplatin chemotherapy, which might suppress the proliferation of damaged cells. This phenomenon was also observed by Setua et al.,32 who reported that cells could recover from damages in GNPenhanced radiotherapy; however, cell growth was arrested when drug was loaded on the GNPs. In addition, it should be mentioned that cell recovery might be influenced by the proliferative capacity of different cell lines.48 SPECT/CT Imaging. To investigate whether the probes could specifically target tumors and to optimize the time point for applying thermotherapy, we examined tumor-bearing mice treated with RGD-125IPt-PDA@GNRs via SPECT/CT imaging. Tumor-bearing mice were injected intravenously with RGD- 125 IPt-PDA@GNRs, RAD- 125 IPt-PDA@GNRs, or RGD-125IPt-PDA@GNRs probes plus free RGD peptide, at a radiation dosage of 300 μCi. SPECT/CT imaging revealed that 10409

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Figure 6. Thermal and chemo-thermal treatment of tumor cells. (A) Immunofluorescence images of H1299 cells incubated with RGDIPt-PDA@GNRs at a concentration of 50 μg Au/mL for 2 h with exposure to an 808 nm laser light for different durations. (B, C) Viability of H1299 cells immediately after treatment (B) or at different maintenance durations (C). Scale bars: 20 μm, **, p < 0.01. Figure 7. In vivo behavior of the probes. (A) Single-photon emission computed tomography/computed tomography imaging of tumor-bearing mice after intravenous injection with RGD-125IPtPDA@GNRs, RAD-125IPt-PDA@GNRs, or RGD-125IPt-PDA@ GNRs probes plus free RGD peptide (100 μL, 0.15 mM), at a radiation dose of 300 μCi. (B) Biodistributions of the probes 6 h after injection. **, p < 0.01. (C) Blood clearance profile of RGD-125IPt-PDA@GNRs after intravenous injection.

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tumor accumulation of RGD- IPt-PDA@GNRs was significant 1 h post-injection, and the radioactive signals in the area of the tumor gradually increased with time and culminated 6 h post-injection (Figure 7A). However, in the presence of competing free RGD peptide, the radioactive signal was reduced, and the tumor accumulation of RAD-125IPt-PDA@ GNRs was marginal. After SPECT/CT imaging, the mice were sacrificed, and the biodistributions of probes in major organs were determined according to the percentage of injected dose per gram of tissue (%ID/g) (Figure 7B). Similar to other nanomaterials,17,49 both RGD-125IPt-PDA@GNRs and RAD-125IPt-PDA@GNRs mainly accumulated in the liver (19.42 ± 5.57% ID/g and 20.33 ± 4.37% ID/g, respectively) and spleen (20.18 ± 4.40% ID/g and 15.29 ± 1.67% ID/g). Tumor accumulation of RGD-125IPtPDA@GNRs was 6.89 ± 0.21% ID/g and decreased to 0.81 ±

0.22% ID/g (p < 0.01) after the addition of free RGD peptide. Tumor accumulation of RAD-125IPt-PDA@GNRs was 2.13 ± 1.76% ID/g, which was significantly lower than that of RGD-125IPt-PDA@GNRs (p < 0.01). These data confirmed the SPECT/CT observations and indicated that the RGD-125IPt-PDA@GNRs indeed specifically targeted the αvβ3 integrin in vivo.39 High accumulations and long-term retention of nanoparticles in the reticuloendothelial system (RES) may induce potential 10410

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Figure 8. Photoacoustic imaging of tumors after intravenous injection with RGD-IPt-PDA@GNRs (RGD) at a dose of 40 mg Au/kg b. w., and RAD-IPt-PDA@GNRs (RAD) as a control. Scale bar = 10 μm.

αvβ3 integrin (CD61) was indeed highly expressed in the tumor angiogenic vessels, and these vessels were well targeted by RGD-IPt-PDA@GNRs (Figure S6). Chemo-Photothermal Combined Therapy and Histological Studies of Tumors. For chemo-photothermal therapy of tumors, mice bearing H1299 tumors (approximately 60 mm3 in volume) were divided into six groups with 10 mice in each group: PBS + laser, free cisplatin + laser, RGD-I-PDA@GNRs, RGD-IPt-PDA@GNRs, RGD-I-PDA@GNRs + laser, and RGD-IPt-PDA@GNRs + laser. The dose of free cisplatin was 2.5 mg/kg body weight (b. w.) in the dose range for human clinical treatment (1.2−2.7 mg/kg b. w.),59 but much less than the nonlethal dose for murine tumor treatment used at the laboratory (8 mg/kg b. w.).30 The dose of the probes was 40 mg Au/kg b. w. containing an equivalent dose of free cisplatin. Tumors were irradiated with a NIR light (0.5 W/cm2, 808 nm) 6 h post-injection. A NIR camera was used to capture the temperature change in the tumor area during thermal therapy (Figure 9). The temperature in the tumor area increased with increased irradiation time. After treatment for 5 min, the temperatures in the tumor area were 49.7 °C, 50.5 °C, 35.8 °C, and 36.6 °C for the mice injected with RGD-IPt-PDA@GNRs,

long-term toxicity, hampering their clinical use.50 Surface modification of nanoparticles with highly hydrophilic and antifouling materials, such as PEG and zwitterionic substances, is effective to evade RES uptake and prolong their blood circulation time.51 Currently, most of gold-nanoformulated drugs in preclinical or clinical trials, such as Aurimmune, Auroshell, and AuNPs, are coated with PEG.52 However, nanoparticles with sizes greater than kidney filtration thresholds (around 5.5 nm) are not renal clearable and are eventually accumulated in and cleared by RES after intravenous injection.53,54 To minimize nonspecific accumulation by RES and potential toxicity in vivo, nanoparticles developed with renal clearance are more desirable, which appears to be the most appropriate way nondegradable nanoparticles are removed from the body because it is the most rapid.55,56 We also evaluated the pharmacokinetic properties of RGD-125IPt-PDA@GNRs (Figure 7C). The blood level of the probes decreased gradually with time and remained over 5% ID/g at 6 h post-injection. By applying a two compartment model,56 the blood clearance half-life of the probes was determined to be 38.15 min. Photoacoustic Imaging of Tumor Region. Angiogenesis is an essential step for the growth and spread of malignant tumors.57 The αvβ3 integrin, a cell adhesion molecule, is a specific marker of angiogenesis, which is highly expressed on activated and proliferating endothelial cells, but generally not on quiescent endothelial cells.58 As previously demonstrated, RGD-125IPt-PDA@GNRs could specifically target αvβ3-positive tumor cells and accumulate in the tumors. Subsequently, we examined the probes located in the tumor angiogenic vessels using high-resolution photoacoustic imaging (PAI). To this end, the tumor-bearing mice were injected intravenously with RGD-IPt-PDA@GNRs (40 mg Au/kg), using RAD-IPt-PDA@ GNRs and PBS as the controls. As indicated in Figure 8, RGDIPt-PDA@GNRs highlighted blood vessels and revealed the detailed vascular structure of the tumors. However, PAI signal intensity was not augmented in the blood vessels of mice receiving RAD-IPt-PDA@GNRs or PBS. These observations confirmed that RGD-IPt-PDA@GNRs specifically addressed tumor angiogenic vessels.40 To further verify these observations, we analyzed the tumor tissues from treated mice with immunohistochemistry using antibodies against αvβ3 integrin and silver staining to detect GNRs. As shown in Figure S5, the

Figure 9. NIR photothermal imaging of tumor-bearing mice during photothermal therapy after intravenous injection with PBS, free cisplatin (2.5 mg/kg), RGD-I-PDA@GNRs, or RGD-IPt-PDA@ GNRs (40 mg Au/kg b. w.) 6 h post-injection. 10411

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Figure 10. Chemo-photothermal combined tumor therapy. (A) Tumor growth curves after different treatments. (B) Representative photographs of tumors after treatment. Scale bar: 1 cm. (C) Hematoxylin/eosin staining of tumor tissues after treatment. Scale bar: 100 μm.

effect, while thermal therapy alone did not. A possible explanation was that NIR irradiation might damage and even kill tumor cells. The proliferation and self-repair of the damaged cells were suppressed by the drug.10 Moreover, in the dead cells, the cell membrane broke down, and the released cisplatin was permeably uptaken by the surrounding tumor cells, leading to increased DNA damage and apoptosis, and thus possibly caused the synergistic effect to prevent tumor recurrence. In particular, our probes can specifically target tumor angiogenic vessels, which supply tumor tissue with oxygen and nutrients and are essential for tumor growth. The tumor microvasculature damaged by the targeted therapy would fail to deliver nutrients to the tumor tissues, leading to ischemic necrosis of the tumor. Damage to tumor microvasculature during therapy may thus be a more effective mechanism for tumor suppression than clonogenic cell death itself.60 Our results indicated that cisplatin-loaded, αvβ3 integrin-targeted GNR probes could ablate tumors and prevent tumor relapse with combined chemo-photothermal therapy. Finally, we examined the potential toxic effects of RGD-IPtPDA@GNRs for the mice. Histological analyses of major organs from mice 21 days post-treatment indicated no noticeable organ damages or significant abnormalities (Figure S7). These preliminary results showed that our RGD-IPtPDA@GNRs exhibited no obvious toxic short-term side effects in mice at the dose used in the present study.

RGD-I-PDA@GNRs, free cisplatin, and PBS, respectively. After treatment, the mice were maintained, and the tumor sizes were measured until 21 days after treatment. Compared to the PBS + laser, free cisplatin + laser, and RGD-I-PDA@GNRs groups (which exhibited approximately 41.45 ± 5.7-, 33.67 ± 3.16-, and 34.84 ± 4.82-fold increases in tumor volumes, respectively), the tumor growth in the RGD-IPt-PDA@GNRs group was significantly suppressed, with an approximate 18.14 ± 3.21-fold increase in the tumor volume ( p < 0.01; Figure 10A). In the RGD-I-PDA@GNRs + laser and RGD-IPt-PDA@ GNRs + laser groups, the tumors began shrinking after treatment and were finally eliminated on the third day posttreatment. During all of the observation periods, mice survived without any tumor relapse in the RGD-IPt-PDA@GNRs + laser group. However, tumor relapses were observed in the original lesion regions of 40% of treated mice in the RGD-IPDA@GNRs + laser group (Figure 10B). Subsequently, we performed histological examinations of tumor tissues post-treatment via hematoxylin/eosin staining. The tumor cells grew densely in the groups of PBS + laser, cisplatin + laser, RGD-I-PDA@GNRs, and RGD-IPt-PDA@ GNRs 21 days post-treatment (Figure 10C). In the RGD-IPDA@GNRs + laser and RGD-IPt-PDA@GNRs + laser groups, the tumors became loose and fragile, and the tumor cells atrophied 2 h post-treatment. Twenty-one days postirradiation, the tumors of all the mice in the RGD-IPt-PDA@ GNRs + laser group disappeared, and no obvious tumor masses were observed in the original tumor areas. However, the tumors in the RGD-I-PDA@GNRs + laser group showed relapse. These results are consistent with the results of our in vitro study, which showed that combined chemo-photothermal therapy could inhibit tumor cell viability through a synergistic

CONCLUSION In summary, we developed a robust theranostic platform for combined tumor chemo-photothermal therapy based on PDAcoated GNRs. The PDA coating suppressed the cytotoxicity of 10412

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RGD Peptide Conjugation. For RGD peptide (c(RGDyC)) conjugation, the PEG-modified and drug-loaded GNRs were first activated by sulfosuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1carboxylate (sulfo-SMCC). In brief, a freshly made 0.1 mL of 0.4 mM sulfo-SMCC was added to a 0.2 mL Pt-DOPA@GNRs suspension (5 mg/mL in pH 8.4 PBS). After vortexing for 20 min, the GNRs were retrieved by ultracentrifugation. For RGD peptide conjugation, the activated GNRs were dispersed into 0.25 mL of RGD peptide (c(RGDyC)) solution (10 mM in pH 7.2 PBS). The mixture was vortexed for 1 h and then collected by ultracentrifugation. The final product was designated as RGD-Pt-PDA@GNRs. The efficiency of PEG modification and RGD conjugation was evaluated using the Elleman method by measuring the free sulfhydryl groups in the reaction media before and after PEG modification and RGD conjugation spectrophotometrically.63 Accordingly, the amount of PEG molecules and RGD peptides immobilized on a GNR was calculated. Iodine-125 Labeling. After RGD peptide conjugation, RGD-PtPDA@GNRs were labeled with the radioisotope iodine-125. For this purpose, Na125I solution (200 μCi) was added into 200 μL of RGD-PtPDA@GNRs suspension (5 mg/mL, pH = 7.4). After gently vortexing for a while, the solution was incubated at room temperature for 20 min. Subsequently, radio-thin-layer chromatography (RTLC; AR2000, Bioscan, Washington, DC, U.S.A.) was performed to evaluate the labeling efficiency, and saline (pH = 7.4) was used as the mobile phase. In this system, 125I-labeled RGD-Pt-PDA@GNR remains at the origin, while free 125I migrates with the mobile phase. The labeling efficiency was calculated by dividing the radioactivity retained at the origin to the total radioactivity added. The final product, RGD-125IPt-PDA@GNRs, was purified using 10 kDa MWCO filters (Millipore, Amicon Ultra0.5) and size exclusion chromatography with disposable columns containing Sephadex G-25 medium, using saline as the eluent. In addition, the RGD-Pt-PDA@GNRs were also labeled with nonradioactive iodine using NaI as a precursor under the same conditions used for 125I labeling (RGD-IPt-PDA@GNRs). At the same time, RAD peptide coupled, radioactive, or nonradioactive iodinelabeled Pt-PDA@GNRs were also prepared as control probes (RAD-125IPt-PDA@GNRs, RAD-IPt-PDA@GNRs). Stability of 125I Labeling under Physiological Conditions. The stability of 125I labeling was evaluated by incubating RGD-125IPtPDA@GNRs in fetal bovine serum (FBS) for different periods of time at 37 °C. Detachment of the radioisotope was monitored using an instant thin-layer chromatography silica gel (ITLC-SG) with saline (pH= 7) as the developing agent. Characterizations. The morphology, size, and EDS element mappings of the GNRs were examined using a JEOL 2100F TEM operating at an acceleration voltage of 200 kV. The optical properties of the GNRs were evaluated using UV−vis spectroscopy (Cary 50 Bio, Varian, U.S.A.). The particle size distributions were analyzed using an image analysis program by measuring the diameters of ≥100 GNRs. The zeta potential measurements were performed using a Malvern Zetasizer Nano ZSP instrument. Long-Term Stability of PDA Coating. To study the long-term stability of PDA coating on the GNRs surface, RGD-IPt-PDA@GNRs were dispersed in Dulbecco’s modified eagle’s medium (DMEM, with 10% FBS) cell culture media under normal (pH = 7.4) or acidic (pH = 5.4) conditions at 37 °C. After stirring for 15 days, the probe was collected and examined via TEM. Drug Release. To study the release profile of the loaded cisplatin, 10 mg of RGD-IPt-PDA@GNRs was suspended in 20 mL of phosphate buffer solution at different pH levels (6.0, 7.4, and 8.5), and the suspensions were loaded into the dialysis tubes (1000 Da MWCO, Sangon Biotech, China) stirring at 37 °C. After a fixed time period, 1 mL of dialysis solution was removed, and the same volume of fresh corresponding buffer was added. The amount of released cisplatin was measured via Polarized Zeeman AAS (Z-2000 Series, HITACHI, Japan). Photothermal Conversion Efficiency in Vitro. The photothermal conversion performances of the probes were evaluated using an 808 nm NIR laser. For this purpose, the RGD-IPt-DOPA@GNRs

CTAB and achieved high cisplatin loading efficiency, chelatorfree iodine-125 labeling, and RGD peptide functionalization (RGD-125IPt-PDA@GNRs). RGD-125IPt-PDA@GNRs could specifically target tumor angiogenic vessels and showed an outstanding chemo-photothermal synergistic therapeutic effect on tumors, leading to sufficient tumor ablation and prevention of tumor recurrence.

EXPERIMENTAL SECTION Materials. All chemicals were obtained from commercial suppliers and used without further purification. CTAB (>98.0%), sodium oleate (NaOL, > 97.0%), hydrogen tetrachloroaurate trihydrate (HAuCl4· 3H2O), L-ascorbic acid (BioUltra, ≥ 99.5%), silver nitrate (AgNO3, > 99%), and sodium borohydride (NaBH4, 99%) were purchased from Sigma-Aldrich. All other chemicals were obtained from Sinopharm Chemical Regents (Shanghai, China). All glassware was cleaned using freshly prepared aqua regia followed by washing with copious amounts of water and oven-dried at 70 °C. Synthesis of Gold Nanorods. GNRs were synthesized as previously reported with minor modifications.6 To this end, gold nanoseeds (0.2 mg/mL) were first synthesized.61 Next, the GNR growth solution was prepared by dissolving 3.6 g of CTAB and 0.494 g of NaOL in 100 mL deionized water (50 °C) in a 250 mL Erlenmeyer flask. After cooling to 30 °C, 5 mL of 4 mM AgNO3 was added. The mixture was kept undisturbed at 30 °C for 15 min, after which 100 mL of 1 mM HAuCl4 solution was added. Then the mixture became colorless, and after 90 min of stirring (700 rpm), 0.89 mL of HCl (37 wt % in water, 12.1 M) was added to adjust the pH value of the mixture. After another 15 min of slow stirring (400 rpm), 0.50 mL of 64 mM ascorbic acid was added, and the mixture was vigorously stirred for 30 s. Finally, 0.32 mL of gold seed solution was injected into the growth solution. After another 30 s of vigorous stirring, the resultant mixture was kept undisturbed at 30 °C for 12 h for GNR growth. The final product was isolated by centrifugation at 8000 rpm for 15 min followed by the removal of the supernatant. The numbers of GNRs per milligram were calculated according to previous reports, assuming that GNR is a hemispherically capped cylindrical nanorods:62 As the aspect ratio Y = L/D, the volume of one gold nanorod (V0) is

V0 =

⎤ πD 3 ⎡ 4 ⎢ + 2(Y − 1)⎥⎦ 8 ⎣3

(1)

where L and D are the length and diameter of the GNR, respectively. The weight of GNRs can be calculated using the formula M = ρV, where ρ and V are density and volume of the GNRs, respectively. Then, the amount (m) of GNRs per milligram is calculated by

m=

V 8M = 4 V0 πD3⎡⎣ 3 + 2(Y − 1)⎤⎦ρ

(2)

Polydopamine Surface Coating. One mg of GNRs was suspended in 1 mL of dopamine solution (2 mM) buffered to pH 8.5 using 10 mM tris (hydroxymethyl) aminomethane buffer. The suspension was sonicated for 20 min, and then the GNRs were collected by centrifugation. The PDA-coated GNRs (PDA@GNRs) were washed with deionized water twice. PEG Modification and Cisplatin Loading (Pt-PDA@GNRs). For the PEG modification, 1 mg of PDA@GNRs was dissolved in 0.2 mL of aqueous HS-PEG-NH2 (MW = 5000 Da) solution (20 mg/ mL). After 2 min of vortexing and overnight incubation at 4 °C, the GNRs were retrieved by centrifugation and washed with deionized water twice. For cisplatin loading, the PEGylated PDA@GNRs (1 mg) were suspended in an aqueous solution of cisplatin (0.3 mL) with different concentrations and stirred for 12 h at room temperature. To calculate the loading efficiency, the drug-loaded GNRs were dissolved in aqua regia, and the concentrations of Pt (CPt) and Au (CAu) were measured via Polarized Zeeman AAS (HITACHI, Z-2000 Series). The amount of cisplatin loaded was normalized to Pt in 1 mg of gold. 10413

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ACS Nano were suspended in glass vials (1 mL) at 12.5 μg/mL and irradiated with the laser at different power settings (2, 3, 5, and 7 W/cm2) or at different concentrations (1.73, 3, and 11 μg/mL) and irradiated at 2 W/cm2. The temperatures of the suspensions were recorded by a thermocouple thermometer (Hi-Tech Optoelectronics Co., Beijing, China) with an accuracy of ±0.1 °C. To examine whether heating could damage the morphology of the GNRs, the RGD-IPt-PDA@ GNRs (12.5 μg Au/mL) were examined via TEM before and after irradiation at 7 W/cm2 for 10 min. Cell Culture. H1299 cells were maintained in DMEM (Gibco, U.S.A.) supplemented with 10% FBS, 2 mM glutamine, penicillin/ streptomycin, 1.2 mM sodium pyruvate, and 1.8 g/L sodium bicarbonate in uncoated tissue culture dishes at a density of 7−9 × 103/cm2 in a standard humidified 5% CO2 and 37 °C incubator. The cells were passaged when they reached 80% confluence. Specificity of the Probes for αvβ3 Integrin. To investigate the specificity of the probes for αvβ3 integrin, the H1299 cells were seeded on cover glass slices and cultured in 6-well plates. After the cell growth reached 75% confluence, the culture medium was replaced by the fresh medium containing RAD-IPt-PDA@GNRs, RGD-IPt-PDA@GNRs, or RGD-IPt-PDA@GNRs plus free RGD peptides (10 μM) at a concentration of 50 μg Au/mL for 1 h. After incubation, the slides were washed three times with PBS (pH = 7.4, 10 mM), stained with DAPI, and then examined via two-photo fluorescence microscopy (A1plus, Nikon, Japan). Horizontal z-stacks were obtained by capturing images with a 0.45 μm interslice distance along the z-axis. To quantify the intracellular gold contents, H1299 cells were seeded in 6-well plates and incubated with the probes under exactly the same aforementioned conditions. After incubation, the cells were washed with PBS (pH = 7.4) three times and digested with aqua regia. The intracellular gold contents were measured via Polarized Zeeman AAS (Z-2000 Series, HITACHI, Japan). Triplet experiments were performed, and the average value was calculated and expressed as mean ± SD in pictograms of gold per cell. Chemotherapy in Vitro. To evaluate the chemotherapeutic effects of the probes, H1299 cells were treated with RGD-IPt-PDA@GNRs, using RGD-I-PDA@GNRs and pristine GNRs as controls, at different concentrations (10, 25, 50, and 100 μg Au/mL) for 6 h or at 50 μg/ mL for different durations. Next, the cell viability was evaluated using the CCK-8 assay (Dojindo, Japan) according to the manufacturer’s instructions. In brief, H1299 cells were planted in 96 multiwell plates at a density of 1 × 104/well. After incubation, the culture medium was removed, and the cells were washed with PBS (pH = 7.4) twice. Subsequently, 10 μL of CCK-8 assay reagent and 90 μL of fresh culture medium were mixed into each well. After a 2 h incubation period, the absorbance of the plates was estimated using the Wallace 1420 multilabel counter VICTOR3 (PerkinElmer, U.S.A.) at 450 nm. Cell viability was expressed as a percentage of the absorbance of cells incubated with the probes to that of cells maintained in the normal culture medium. To further verify the chemotherapeutic effects, after incubation with the probes at a concentration of 50 μg/mL for 36 and 72 h, the cells were stained against γ-H2AX, using free cisplatin as a positive control. To this end, the treated cells were first fixed by paraformaldehyde. For staining, the fixed cells were blocked with 10% goat serum made in 0.1% Triton X-100 in PBS and incubated with rabbit polyclonal anti-γH2AX antibody (1:300, Invitrogen) at 4 °C overnight. After incubation, the cells were washed three times with PBS (pH = 7.4) and stained with Alexa-488 conjugated secondary antibody (1:500, Invitrogen) for 2 h at room temperature. The nuclei were counterstained with DAPI. Thermal Therapy in Vitro. To evaluate the thermal therapeutic effect of the probe, the H1299 cells were incubated with RGD-IPtPDA@GNRs (50 μg Au/mL) for 2 h. After incubation, the cells were washed twice with PBS (pH = 7.4) and supplemented with fresh culture medium. Subsequently, the cells were treated with the NIR laser (808 nm, 2 W/cm2) for different periods of time (5, 10, and 15 min), stained with calcein AM (2 μM) and PI (4 μM) to indicate the live and dead cells, and examined by confocal laser scanning

microscopy (Leica, Germany), successively. The cell viability after NIR laser exposure was also examined using the CCK-8 assay. Combined Chemo-Photothermal Therapy on Tumor Cells. To demonstrate the effects of combined chemo-photothermal therapy of the probes on tumor cells, H1299 cells were seeded in 96-well plates and incubated with RGD-IPt-PDA@GNRs using RGD-I-PDA@GNRs as a control at a concentration of 50 μg Au/mL for 2 h. After incubation, the cells were washed twice with PBS (pH = 7.4), replenished with fresh culture medium, and treated with the NIR laser (808 nm, 2 W/cm2) for 10 min. After laser treatment, the cells were maintained for different periods of time, and cell viability was evaluated using the CCK-8 assay. SPECT/CT Imaging and Biodistribution of the Probes. All the animal experiments were approved by the Animal Protection and Care Committee of Shanghai Jiao Tong University. Balb/c nude mice (Slaccas, Shanghai, China) with an average weight of 20 g were used. H1299 cells (5 × 106 cell/site) were implanted subcutaneously into nude mice and allowed to grow for 4−5 weeks after inoculation. For SPECT/CT imaging, mice were injected with 0.15 mL (300 μCi) of RAD-125IPt-PDA@GNRs, RGD-125IPt-PDA@GNRs, or RGD-125IPtPDA@GNRs + free RGD peptides (100 μL, 0.15 mM)39 through the tail vein with five mice in each group. SPECT/CT imaging was performed using a small-animal imaging system with a multipinhole collimator SPECT/CT scanner (Nano SPECT/CT PLUS, Bioscan, USA) at 1, 3, 6, and 24 h post-injection. After SPECT/CT imaging, the mice were sacrificed, and the biodistributions of the probes were investigated. For this purpose, the major organs (heart, liver, spleen, lungs, kidney, stomach, intestine, and muscle) and tumor tissues were surgically removed and weighed in plastic test tubes. The radioactivity was determined in a well-type scintillation detector along with 3 × 0.5 mL aliquots of the diluted standard representing 100% of the injected dose. The mean activities were used to obtain the percentage of injected dose per gram of tissue (% ID/g).64 Blood Half-Life. To determine the blood half-life of the probes, H1299 tumor-bearing mice (approximate tumor volume of 300 mm3, n = 5) were intravenously injected with RGD-125IPt-PDA@GNRs (150 μL, 100 μCi). The blood samples were obtained from the tail vein using capillary tubes at different time points (10, 15, 20, 30, 60, 120, and 240 min) after injection and counted by a well-type scintillation detector to obtain a time−activity blood curve. Half-life of RGD-125IPt-PDA@GNRs in %ID/g was fitted to a two-compartment bolus intravenous injection model50 and determined using the biexponential nonlinear regression of graphing software (GraphPad prism 6.0, GraphPad Software). Photoacoustic Imaging. To examine whether the probes targeted tumor angiogenic vessels, high-resolution photoacoustic imaging (PAI) of tumors was performed. Mice bearing H1299 tumors were intravenously injected with 40 mg Au/kg b. w. of RGD-IPt-PDA@ GNRs using RAD-IPt-PDA@GNRs and PBS as controls. An experimental laser-scanning optical-resolution photoacoustic microscopy (LSOR-PAM) system was used for imaging.65 This system has a pulsed Nd:YAG laser with a 532 nm wavelength and a repetition rate of 10 kHz, which allows us to utilize the transverse LSPR absorption of the GNRs for PAI. The lateral and axial resolutions of the system are 5 and 50 μm, respectively. PAI images were acquired at 1, 3, and 6 h after injection. Immunohistochemistry and Histological Studies. After PAI, the mice were sacrificed, and the tumors were obtained to perform immunohistochemical staining against h integrin (CD 61) and CD31 and silver staining for GNRs. For immunohistochemical staining, tumors were fixed in paraformaldehyde, embedded in paraffin, and then sectioned. The tumor slices (10 μm thick) were mounted on glass slides, dewaxed in xylene, dehydrated in ethanol, and then incubated in hydrogen peroxide (3%) for 10 min. After the antigen was retrieved by microwave treatment, the sections were first incubated with a 1:100 dilution of anti-CD61 antibody (rat antimouse, BD Biosciences, San Diego, California) overnight at room temperature and then incubated for 1 h with a biotinylated goat antirat IgG in combination with streptavidin-HRP and the DAB detection system. Finally, the tumor sections were counterstained with hematoxylin and 10414

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ACS Nano returned to ammonia solution. The procedure for CD31 staining was similar to that for CD61 staining; however, anti-CD31 primary antibody (rat antimouse, BD Biosciences, San Diego, California) was used. For silver staining, tumors were embedded in OCT (Miles, Elkhart, IN), frozen on dry ice, and sectioned into 12 μm-thick slices using a cryostat (CM 1900 UV, Leica, Germany). The slices were fixed with paraformaldehyde (4%) for 5 min and washed thrice with PBS (pH 7.4). Silver staining was performed by incubating the fixed slices in a mixture of ProteoSilver Silver Solution (Sigma-Aldrich) and sodium thiosulfate solution (2.5%, weight ratio) for 5 min successively. Slices were washed carefully and evaluated by light microscopy, and cells exhibiting brown intracellular particles were considered positively stained. Tumor Therapy. When the tumors grew to approximately 60 mm3 in volume, the mice were divided into six groups with 10 mice in each group as follows: PBS + laser, free cisplatin + laser, RGD-I-PDA@ GNRs, RGD-IPt-PDA@GNRs, RGD-I-PDA@GNRs + laser, and RGD-IPt-PDA@GNRs + laser. The free cisplatin and probes were intravenously injected at a dose of 2.5 mg/kg and 40 mg Au/kg b. w., respectively. The drug-loaded probes administered contained equivalent doses of free cisplatin. For thermal therapy, the tumors were irradiated using a NIR laser light (0.5 W/cm2, 808 nm) 6 h postinjection. After treatment, the tumor sizes were measured using a caliper every 2 days for 21 days. The tumor volumes (V, mm3) were calculated using the formula V = 3πab2/4,66 where a and b refer to the length and width of the tumor, respectively. Relative tumor volumes were calculated as V/V0 (V0, tumor volume before treatment). To evaluate the therapeutic effect and biocompatibility of the probes, tumors and major organs (heart, liver, spleen, lungs, kidneys, and tumor) were surgically removed after the death of mice during the experimental period or at the end of the treatment after sacrifice. The tissues were fixed in paraformaldehyde and embedded in paraffin, sectioned into 12 μm-thick slices, and stained with hematoxylin/eosin.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b06267. Zeta potentials of the pristine GNRs and the modified GNRs; UV−vis spectra of thiol groups in PEG and RGD peptide before and after PEG modification and RGD peptide conjugation analyzed using Elleman assay method; TEM and HRTEM images of RGD-IPtPDA@GNRs after 15 days of votexing in the cell culture medium (pH = 5.4); the photothermal conversion performances and morphology of RGD-IPt-PDA@ GNRs; γ-H2AX immunostaining of H1299 cells; immunohistochemical staining of tumor vasculatures; silver staining of the tumor tissues after PAI; hematoxylin/eosin staining of mouse major organs (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Lu Zhang: 0000-0001-7049-0760 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (Grants 81230030 and 81571729) and grants from the State Key Laboratory of Oncogenes and Related Genes (90-15-03). 10415

DOI: 10.1021/acsnano.6b06267 ACS Nano 2016, 10, 10404−10417

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