pH-Dependent Transmembrane Activity of Peptide-Functionalized

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pH-Dependent Transmembrane Activity of Peptide-Functionalized Gold Nanostars for Computed Tomography/Photoacoustic Imaging and Photothermal Therapy Ying Tian,† Yunlei Zhang,† Zhaogang Teng,†,‡ Wei Tian,† Song Luo,† Xiang Kong,† Xiaodan Su,§ Yuxia Tang,† Shouju Wang,*,† and Guangming Lu*,†,‡ †

Department of Medical Imaging, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, People’s Republic of China ‡ State Key Laboratory of Analytical Chemistry for Life Science School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, People’s Republic of China § Key Laboratory for Organic Electronics & Information Displays and Institute of Advanced Materials, Nanjing University of Posts and Telecommunications, Nanjing 210046, People’s Republic of China S Supporting Information *

ABSTRACT: Progress in multifunctional nanomaterials for tumor therapy mostly depends on the development of tumortargeting delivery strategies. One approach is to explore a pHresponsive strategy to target the slightly acidic solid tumor microenvironment. A novel class of pH (low) insertion peptides (pHLIPs) with pH-dependent transmembrane activity can fold and rapidly insert into the lipid bilayer of tumor cells triggered by acidity, facilitating the cellular internalization of nanomaterials synchronously. Here, we innovatively decorated gold nanostars (GNSs) with pHLIPs (GNS-pHLIP) to improve their targeting ability and photothermal therapeutic (PTT) efficiency. The obtained GNS-pHLIP exhibited the excellent characteristics of uniform size and good biocompatibility. As compared to GNS-mPEG, the cellular internalization of GNS-pHLIP was 1-fold higher after a 2 h incubation with cells in media at pH 6.4 than at pH 7.4. Moreover, the tumor accumulation of the GNS-pHLIP was 3-fold higher than that of GNS-mPEG after intravenous injection into MCF-7 breast tumor animal models for 24 h. Furthermore, GNS-pHLIP exhibited stronger signals than the GNS-mPEG through computed tomography (CT) and photoacoustic (PA) imaging. Simultaneously, the desirable targeting efficiency significantly improved the PTT efficacy to tumors, with low side effects on normal tissues. The results clearly demonstrate that the GNS-pHLIP successfully took advantage of the tumor-targeting ability of pHLIPs and the good characteristics of GNSs, which may contribute to the study of tumor imaging and therapy. KEYWORDS: pH-responsive strategy, gold nanostars, pH (low) insertion peptides, computed tomography, photoacoustic imaging, photothermal therapy

1. INTRODUCTION

enhanced permeability and retention (EPR) effects, which is classified as passive targeting. Although the EPR effect is widely applied, the efficacy of this effect is always affected by the tumor microenvironment, such as blood flow and stroma changes and tumor heterogenicity.8,9 In addition to the passive transport strategy, nanomaterials can increase their accumulation in tumor sites by physically absorbing or covalently conjugating antibodies or targeting ligands for active targeting.10,11 However, biological antibodies or ligands are always constrained to a single target; thus this active targeting strategy is greatly influenced by the heterogeneity of tumors and the

The development of multifunctional agents that could be used for achieving tumor imaging and treatment simultaneously has become a major focus of tumor research.1,2 In recent years, multifunctional nanomaterials have made a great contribution to the development of such agents that have the perfect combination of diagnostic and therapeutic effects on a single nanoplatform.3−5 However, the currently limited tumor retention and low delivery efficiency of such nanomaterials often restrict their clinical translation.6,7 Thus, targeted delivery strategies should be highlighted and designed to improve the tumor-targeting efficiency of these nanomaterials. At present, multifunctional nanomaterials mainly depend on their surface properties to transport and penetrate through irregular tumor vessels to target tumor sites mediated by © XXXX American Chemical Society

Received: October 23, 2016 Accepted: January 6, 2017 Published: January 6, 2017 A

DOI: 10.1021/acsami.6b13237 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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2. MATERIALS AND METHODS

dynamic changes in the corresponding receptors, leading to inaccuracies in tumor imaging and treatment.12,13 One alternative approach has been explored to develop pHresponsive strategies based on the specific acidosis character of solid tumor microenvironments.14 As compared to the neutral surroundings of normal organs (pH 7.4), solid tumor tissues are more acidic, with pH values of 6.0−7.0. In addition, the acidic extracellular microenvironment of tumors is related to abnormal glycolysis and hypoxia of tumor cells.15 Many efforts have been made to develop pH-responsive nanomaterials through destabilization or degradation of acid-liable linkers between nanoparticles and agents by acid stimulation.16 To date, the development of precise pH-responsive nanosystems is still a challenge when faced with the problem of biocompatibility or the functional changes in nanomaterials after modification. Recently, 35-amino acid peptides with pH-dependent transmembrane activity called pH (low) insertion peptides (pHLIPs) have been recognized.17−19 At neutral pH, pHLIPs are water-soluble in an equilibrium state. When in a low-pH environment, the aspartate or glutamate amino acid residues from the pHLIPs are protonated, increasing the hydrophobicity of the peptides. As a result, pHLIPs activated by acidity can rapidly insert into the lipid bilayer of tumor cells and form a stable transmembrane α helix, thus facilitating the entry of nanomaterials attached to their C-terminal tails into the cells.20,21 Moreover, pHLIPs are sensitive only to acid stimulation, not neutral surroundings, thus reducing the side effects on normal tissues. Although pHLIPs exhibit excellent pH response effects, they should be further explored for tumor theranostic applications because of their lack of intrinsic antitumor and imaging activities.22,23 Therefore, the modification of multifunctional nanomaterials with pHLIPs will synergistically enhance their significant properties to achieve high efficiency in tumor-targeting imaging and treatment. Here, we innovatively decorated anisotropic gold nanomaterials, gold nanostars (GNSs) with pHLIPs, and attempted to develop tumor theranostics. GNSs are small, have low toxicity, are plasmon tunable in the NIR region, are simple to synthesize, and exhibit excellent biocompatibility and effective photothermal therapy (PTT).24−26 In addition, GNSs have been widely explored as contrast agents for computed tomography (CT) and photoacoustic (PA) imaging. Therefore, these materials are regarded as suitable candidates for multifunctional diagnosis and imaging agents.27 This innovative strategy will boost the efficiency of tumor targeting and realize CT/PA imaging-guided enhanced PTT in a more favorable and simple system. As compared to bare GNSs, GNSs modified with pHLIPs (GNS-pHLIP) showed 1-fold higher cellular internalization when incubated with MCF-7 breast tumor cells in pH 6.4 media for 2 h in vitro. Moreover, intravenous injection of GNS-pHLIP into animal models caused 3-fold higher tumor accumulations after 24 h and significant tumor suppression after NIR PTT in vivo. In addition, the in vivo tumor-targeting ability of the nanomaterials was accurately monitored using CT/PA imaging. Therefore, GNS-pHLIP targeted the acidic solid tumor microenvironment and exhibited excellent PTT effects under precise CT/PA imaging guidance, which may have great potential value in tumor imaging and therapy in the near future.

2.1. Materials. Sodium citrate, chloroauric acid (HAuCl4·3H2O), ascorbic acid, and AgNO3, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Shanghai, China). The protein assay dye reagent was purchased from BIO-RAD (Hercules, CA). Methoxy-PEG-thiol (mPEG-SH, MW ≈ 1000 Da) and amine-PEG-thiol (NH2-PEG-SH, MW ≈ 1000 Da) were purchased from Laysan Bio. Inc. (Shanghai, China). Human breast tumor cells (MCF-7) were purchased from ATCC (Manassas, VA). Fetal bovine serum (FBS), trypsin, dry powder of Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffered saline (PBS), and penicillin/streptomycin were purchased from Gibco/Life Technologies (Shanghai, China). Specifically, DMEM solutions were from dry powder with 3.7 g/L NaHCO3 (pH 7.4) or 0.37 g/L NaHCO3 and 2.31 g/L NaCl (pH 6.4). Maleimide-PEG2000-pHLIPs (AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG) were synthesized and purified by Nanjing Leon Biological Technology Co. Ltd. (Nanjing, China). 2.2. Synthesis of GNS-mPEG and GNS-pHLIP. GNSs were synthesized by a previously reported method.28,29 First, gold seeds were prepared by adding 3 mL of 1% sodium citrate solution to 100 mL of boiling 1.0 mM HAuCl4 solution, and the solution color turned from colorless to dark red under vigorous stirring followed by cooling to room temperature. Next, 100 μL of gold seed was added to 10 mL of 0.25 mM HAuCl4 solution under vigorous stirring, followed by the simultaneous addition of 50 μL of 0.1 M ascorbic acid and 40 μL of 0.01 M AgNO3. To obtain PEGylated GNSs, 20 μL of mPEG-SH (2 mM) was added to the bare GNS solution and continuously stirred for 1 h. The product was purified by centrifugation and termed GNSmPEG. To obtain pHLIP-functionalized GNSs, 10 μL of 2 mM NH2PEG-SH was mixed with 10 μL of 2 mM maleimide-PEG2000-pHLIPs for 30 min and then added to the bare GNS solution for another 1 h under stirring. The product was purified by centrifugation and termed GNS-pHLIP. 2.3. Characterization of the GNS-mPEG and GNS-pHLIP. The sizes and morphologies of the GNS-mPEG and GNS-pHLIP were tested using a JEOL JEM-2100 (Japan) transmission electron microscope (TEM), and the corresponding absorbance spectra were detected using a UV/vis spectrometer (Lambda 35, PerkinElmer, U.S.). The diameters and surface charges of the GNS-mPEG and GNS-pHLIP were characterized with a ZetaPALS analyzer (Brookhaven, U.S.). To test the stability of GNS-pHLIP, nanoparticles were dispersed in different solutions including FBS, media with pH values of 6.4 and 7.4, and then analyzed by a UV/vis spectrometer and ZetaPALS analyzer. 2.4. CT and PA Imaging Measurement of the GNS-pHLIP. For phantom CT imaging, different concentrations (0.5, 2, 4, and 10 mg/ L) of GNS-pHLIP were imaged by a clinical dual-source CT system (Somatom Definition, Siemens Healthcare, Forchheim, Germany) and quantified by a dedicated workstation (MultiModality Workplace, Siemens Medical Solutions, Erlangen, Germany). For phantom PA imaging, signals of GNS-pHLIP solutions were imaged using a photoacoustic 3-D tomographic imaging system (Endra Nexus 128, U.S.) at an absorption wavelength of 808 nm. The signal intensity was analyzed by OsiriX Lite software. 2.5. Photothermal Property Measurement of the GNSpHLIP. Different concentrations (0.5, 1, 2, and 4 mg/L) of GNSpHLIP were irradiated with an 808 nm wavelength laser (Hi-Tech Optoelectronics Co., Ltd., China) at a power density of 1 W/cm2. In addition, 2 mg/L GNS-pHLIP was irradiated at different power densities (0.2, 0.5, 1, and 1.5 W/cm2) for 5 min to test the temperature variation. To test the impact of acidity on the temperature, 2 mg/L GNS-pHLIP was dissolved in media at pH 7.4 and pH 6.4 and then irradiated for 5 min. To investigate the photostability, GNS-pHLIP (2 mg/L) was exposed to the laser at a power density of 1 W/cm2 for five ON−OFF cycles (ON, 3 min; OFF, 10 min). The morphology of the GNS-pHLIP before and after irradiation then was imaged by TEM, respectively. The corresponding temperature changes during laser irradiation were monitored in real time using an infrared camera B

DOI: 10.1021/acsami.6b13237 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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2.13. Statistical Analysis. Data analyses were conducted using the software GraphPad Prism 6.0. Comparisons between the two groups were performed using Student’s test, and the statistical analyses among multiple groups were performed using two-way ANOVA. A p value less than 0.05 was considered statistically significant.

(MAGNITY f15F1, Wuhan VST Light & Technology Co., Ltd., China). 2.6. Cell Culture and Cellular Uptake Quantification Analysis. Breast tumor cells (MCF-7) were all cultured in DMEM with 1% penicillin/streptomycin and 10% FBS in a humidified incubator at 37 °C under 5% CO2 atmosphere. When the confluence of the cells reached 70%, cells were incubated with GNS-mPEG (2.5 mg/L) and GNS-pHLIP (2.5 mg/L) at pH 6.4 or 7.4 for 2 h. The cells were then collected and redispersed in 3 mL of PBS. After, 400 μL cell suspensions were used to quantify the protein concentration using a Bradford assay. The remaining was digested with aqua aqua regia and redissolved in 2% HNO3. The concentration of gold was then tested using inductively coupled plasma (ICP) measurements. The uptake of gold was analyzed as the weight ratio between the gold and protein. The cellular uptake of GNS-pHLIP was also visualized by bright-field microscopic and TEM imaging. 2.7. Cell Viability Assay. Approximately 1 × 104 cells were cultured on 96-well plates when the cells concentration reached 70%. Cells then were incubated with GNS and GNS-pHLIP with a concentration gradient of 0, 0.5, 1, 2, and 4 mg/L in DEME (pH 6.4/ 7.4) without irradiation. After the cells were incubated for 4 h, they were washed with PBS three times, and their viabilities were tested by MTT assays. For the PTT effect evaluation, MCF-7 cells were treated as described above after incubation for 4 h. The cells then were exposed to an 808 nm wavelength diode-pumped solid-state NIR laser system for 3 min. In addition, the PTT efficiency was measured using an MTT assay after laser irradiation. 2.8. Tumor Accumulation of the GNS-mPEG and GNS-pHLIP. Approximately 5 × 106 MCF-7 cells were collected to subcutaneously inject into the right flank of nude mice to establish breast tumor animal models. Mice (n = 12) were chosen randomly for intravenous injection with 100 μL of 25 mg/L GNS-pHLIP or GNS-mPEG. Tumors were dissected and weighed 12 and 24 h postinjection, digested with aqua regia, and redissolved in 5 mL of 2% HNO3. The collections were quantified to measure the concentration of gold by ICP. The gold amounts were calculated as the ratio of microgram of gold per gram of organ according to the above results. 2.9. CT/PA Imaging of Xenograft Models. Tumor-bearing mice (n = 6) were injected intravenously with 100 μL of 25 mg/L GNSmPEG or GNS-pHLIP. For CT imaging in vivo, mice were anaesthetized and scanned using a clinical dual-source CT system at 0, 1, 4, 12, and 24 h postinjection. The CT attenuation values (Hounsfield units, HU) were tested by a dedicated workstation. The PA imaging was performed before and 24 h after injection of the GNSmPEG and GNS-pHLIP using a photoacoustic 3-D tomographic imaging system (Endra Nexus 128, U.S.) at an absorption wavelength of 808 nm. 2.10. Photothermal Effect Evaluation in Vivo. Tumor-bearing mice (n = 3) were injected intravenously with 100 μL of PBS or with 25 mg/L GNS-mPEG or GNS-pHLIP. The mice were anaesthetized to irradiate using an 808 nm wavelength NIR laser at a power density of 2 W/cm2 for 4 min at 24 h postinjection. The temperature of the tumors was monitored using a MAGNITY f15F1 infrared camera. 2.11. PTT Efficacy in Vivo. Tumor-bearing mice were divided randomly into six groups (n = 6/group). Three groups were injected intravenously with 100 μL of PBS or with 25 mg/L GNS-mPEG or GNS-pHLIP and irradiated with a laser at 2 W/cm2 for 4 min at 24 h postinjection. The other three groups were treated equally but without laser irradiation. Body weights and tumor volumes of each group were measured every 3 days. The volume of the tumor was measured by a caliper and calculated according to the following equation: volume = (tumor greatest longitudinal diameter) × (tumor greatest transverse diameter)2 × 0.5. In total, 57 mice were used for the test in vivo. 2.12. Histopathology. Tissue specimens, including the liver, heart, spleen, kidney, lung, and tumor, were dissected and fixed with 10% formalin and embedded in paraffin after PTT treatment. Approximately 4 mm thick sections were prepared for pathological sectioning and hematoxylin and eosin (H&E) staining.

3. RESULTS AND DISCUSSION The GNSs were prepared according to a previous protocol.27 Importantly, the GNSs were easy to prepare and free of cetyltrimethylammonium bromide (CTAB) to avoid potential cytotoxicity. Both GNS-mPEG and GNS-pHLIP exhibited stable dispersions and protruding sharp branches with a mean diameter of 60 nm, as shown in the TEM images (Figure 1a).

Figure 1. (a) TEM images of the GNS-mPEG and GNS-pHLIP. Scale bars: 50 nm. (b) UV−vis spectra of the GNS-mPEG and GNS-pHLIP from 500 to 1100 nm. (c) Hydrodynamic diameters of the GNSmPEG and GNS-pHLIP determined by DLS measurements. (d) Zeta potentials of the GNS-mPEG and GNS-pHLIP. (e) UV−vis spectra of the GNS-mPEG and GNS-pHLIP in FBS and DMEM at pH 6.4 and 7.4. (f) Hydrodynamic diameters of the GNS-mPEG and GNS-pHLIP in FBS and DMEM at pH 6.4 and 7.4.

In addition, the UV/vis spectra peaked at approximately 808 nm, with broad width facilitating irradiation within the NIR region (Figure 1b). The hydrodynamic sizes of the GNS-mPEG and GNS-pHLIP were 81.55 ± 0.78 and 85.48 ± 1.29 nm, respectively, as determined by dynamic light scattering (DLS, Figure 1c). The size of the GNS-pHLIP was slightly larger due to the mass coating of the small peptide. The zeta potentials of the GNS-mPEG and GNS-pHLIP exhibited negative changes of −19.02 ± 0.46 and −11.21 ± 1.01 mV, respectively. The discrepancy in the charge indicated the successful binding of pHLIPs (Figure 1d). To investigate the stability of the GNSmPEG and GNS-pHLIP in a physiological environment, both were dispersed in FBS and DMEM at pH 6.4 and 7.4. As shown in Figure 1e and f, no obvious differences appeared in the UV− vis absorption and hydrodynamic size data when the nanoparticles were in pure serum or in the simulated environments for normal tissues (pH 7.4) and tumors (pH 6.4). The DLS C

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Figure 2. (a) CT images and plot of the CT values (HU) of the GNS-pHLIP at different concentrations. The concentrations from 1 to 4 are 0.5, 2, 4, and 10 mg/L, respectively. (b) PA images and plot of the PA intensities of the GNS-pHLIP at 0.5, 2, 4, and 10 mg/L.

Figure 3. (a) Thermal images and temperature curves of GNS-pHLIP at different concentrations with an 808 nm laser at 1 W/cm2 for 5 min. (b) Thermal images and temperature curves of GNS-pHLIP in DMEM at pH 6.4 and pH 7.4 with an 808 nm laser at 1 W/cm2 for 5 min. (c) Thermal images and temperature curves of GNS-pHLIP (2 mg/L) at different laser power intensities.

measurement revealed stable hydrodynamic diameters of GNSpHLIP in DMEM at pH 7.4 and pH 6.4 for at least 48 h (Figure S1). The results show that GNS-pHLIP can be stable in the physiological environment at different pH values. In addition, the zeta potentials of GNS-pHLIP at pH 7.4 and pH 6.4 were −8.05 ± 1.03 and −3.61 ± 2.10 mV, respectively, suggesting the surface charge of GNS-pHLIP was similar at pH 7.4 and pH 6.4. Because of the high X-ray attenuation coefficient and excitation of the surface plasmon resonance (SPR) of the GNS,30 GNS-pHLIP could be used as novel CT/PA contrast agents with high sensitivity and spatial resolution. CT is a widely used imaging modality that is advantageous due to its distinct contrast, high spatial resolution, and isotropic three-

dimensional (3D) imaging capability; however, due to the poor soft-tissue contrast of CT, its sensitivity for detection of some malignant tumors is low.31 PA imaging is a newly emerging and noninvasive modality that simultaneously combines optical and ultrasonic imaging technologies.32 The combination of CT and PA imaging induced by the GNS-pHLIP could be very significant for achieving a more precise diagnosis and better guidance for tumor treatment. To explore the imaging ability of the GNS-pHLIP, it was thoroughly evaluated by CT and PA imaging systems. As shown in Figure 2, it was noted that the CT values and PA intensities of the GNS-pHLIP increased in parallel as the concentration increased and were linearly proportional to the concentration (R2 = 0.97). At a concentration of 10 mg/L, the intensity of the CT and PA D

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0.21 ± 0.06 (ng Au/ng protein), which was significantly lower than that of GNS-pHLIP treated tumor cells at pH 6.4. The low uptake for normal cells further guaranteed the targeting ability of GNS-pHLIP for tumor cells. The in vivo tumor-targeting efficiency of the GNS-pHLIP was explored using the ICP method. As shown in Figure 4b, the accumulation of GNS-pHLIP in the tumor at 12 and 24 h postinjection was 0.77 ± 0.30 μg Au/ng tumor and 1.68 ± 0.44 μg Au/ng tumor, respectively. Moreover, a clear difference was observed between the amount of gold in the tumors (GNSmPEG, 0.98 ± 0.32 μg Au/ng tumor; GNS-pHLIP, 1.68 ± 0.44 μg Au/ng tumor; p = 0.01) at 24 h postinjection. These results confirmed that pHLIPs endowed excellent tumor-targeting abilities to GNSs through a pH-dependent pathway both in vitro and in vivo. This finding is very important for further tumor-targeting imaging and treatment studies. Because biocompatibility is a critical factor for their clinical application, cytotoxicity analysis was carried out by MTT assay by incubating various concentrations of GNS-mPEG and GNSpHLIP with MCF-7 cells at both pH 7.4 and 6.4 for 4 h. As seen in Figure 5, as the concentrations changed from 0.5 to 4

imaging reached 61.37 Hounsfield units (HU) and a 5613.29 photoacoustic intensity. The results suggest the potential value of GNS-pHLIP as next-generation CT/PA contrast agents. To investigate the photothermal property of the GNSpHLIP, the potential influence factors such as concentration, pH value, and laser power intensity were fully evaluated. With an increasing concentration, the temperature of the GNSpHLIP solution increased correspondingly after irradiation with an 808 nm laser (1 W/cm2) for 5 min. At the concentration of 1 mg/L, the temperature reached approximately 42 °C, which was high enough to damage the tumor cells (Figure 3a). To analyze the photothermal conversion efficiency of the GNSpHLIP at different pH values, temperature curves of GNSpHLIP dissolved in DMEM at pH 6.4 and 7.4 were recorded during irradiation. As compared to the medium itself, the temperature of the DMEM containing GNS-pHLIP (2 mg/L) increased rapidly upon laser irradiation at 1 W/cm2 for 5 min, independent of pH values (Figure 3b). The result showed that GNS-pHLIP maintained excellent photothermal conversion in a complex bioenvironment, which is necessary for future use in vitro and in vivo. Moreover, as shown in Figure 3c, the maximum temperature of the GNS-pHLIP solutions was positively correlated with the laser power and reached 85 °C at 1.5 W/cm2. It is also noted that the GNS-pHLIP has excellent photostability, corroborated by its unchanged photothermal conversion ability and morphology after five cycles of laser irradiation (Figure S2). Further, the tumor-targeting and pH-responsive abilities of the GNS-pHLIP were first assessed in vitro using MCF-7 and MCF-10A cells. Tumor cells were incubated with GNS-mPEG and GNS-pHLIP in the media at pH 7.4 or 6.4 for 2 h. As depicted in Figure 4a, the intracellular concentrations of gold

Figure 4. (a) The degree of cellular uptake of GNS-mPEG and GNSpHLIP in vitro at pH 7.4 and pH 6.4 (asterisks indicate p < 0.05). (b) The accumulation of GNS-mPEG and GNS-pHLIP in tumors after intravenous injection into breast tumor animal models for 12 and 24 h (asterisks indicate p < 0.05).

Figure 5. Relative cell viability of the MCF-7 cells after incubation with GNS-pHLIP (a) and GNS-mPEG (b) at different concentrations with and without irradiation (808 nm, 1 W/cm2, 3 min).

from GNS-mPEG-treated cells at pH 6.4 and 7.4 were 0.14 ± 0.03 and 0.19 ± 0.07 (ng Au/ng protein), respectively, while for the GNS-pHLIP-treated cells, they were 0.36 ± 0.09 and 0.68 ± 0.06 (ng Au/ng protein), respectively. The intracellular concentrations of the GNS-pHLIP were approximately 1-fold higher at pH 6.4 than at pH 7.4 (p = 0.02), which possibly benefited from the special properties of pHLIPs. This result was also corroborated by bright-field and TEM images (Figure S3). In contrast, there were no remarkable differences in the intracellular concentrations of gold between the GNS-mPEGtreated cells at different pH values. Moreover, the intracellular concentration of gold in MCF-10A cells (normal human mammary epithelial cells) after 2 h of incubation at pH 7.4 was

mg/L, the cell viability was still more than 80%. This result indicates the excellent biocompatibility and safety of the gold nanomaterials. To investigate the photothermal therapeutic efficiency, MCF-7 cells were incubated with different concentrations of GNS-mPEG and GNS-pHLIP for 4 h and irradiated by an 808 nm wavelength laser (1 W/cm2, 3 min). Figure 5 showed that both the GNS-mPEG and the GNSpHLIP exhibited a concentration-dependent therapeutic photothermal effect. The viability of the cells treated with GNSpHLIP displayed a notable difference between pH 7.4 and pH 6.4. As compared to the cells cultured in the media at pH 7.4, the GNS-pHLIP-treated groups showed a superior PTT effect in acid media (Figure 5a). The MTT results demonstrated that E

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Figure 6. (a) CT images of tumor-bearing mice before and 1, 4, 12, and 24 h after intravenous injection of GNS-mPEG or GNS-pHLIP. (b) The corresponding CT values of tumors from GNS-mPEG- or GNS-pHLIP-treated groups (asterisks indicate p < 0.05). (c) PA images and optical images of mice before and after intravenous injection of GNS-mPEG or GNS-pHLIP for 24 h. (d) The corresponding PA intensities of the tumors from GNS-mPEG- or GNS-pHLIP-treated groups (asterisks indicate p < 0.05).

Figure 7. (a) Thermal images and representative photographs of tumor-bearing mice after laser irradiation for 4 min (808 nm, 2 W/cm2). The red circles indicate the locations of the tumors. (b) The tumor temperatures during the period of irradiation in each group of mice.

obviously indicate that GNS-pHLIP can be used as tumortargeting CT/PA contrast agents to perform precise molecular imaging. To realize the most effective PTT treatment with minimal side effects in vivo, the mice were divided randomly into three groups and intravenously injected with 100 μL of 25 mg/L GNS, GNS-pHLIP, or PBS. Next, the mice were irradiated by an 808 nm wavelength laser at 2 W/cm2 for 4 min after injection for 24 h. The tumor temperature changes were recorded using thermal imaging software during the treatment. As revealed in Figure 7, the tumor temperature of the GNStreated groups increased with increasing laser irradiation. In the GNS-mPEG-treated group, the tumor sites increased by 8 °C after irradiation as compared to the initial temperature. However, there was an approximately 14 °C increase in the group injected with GNS-pHLIP probably due to the higher accumulation of GNS-pHLIP in the tumors. In contrast, the PBS-treated group showed a negligible temperature change. The results demonstrated that the radiation power density and exposure time could sufficiently ensure the PTT efficacy of GNS-pHLIP and kill more tumor cells without obvious damage to normal tissues. To further evaluate the therapeutic efficiency of GNS-mPEG and GNS-pHLIP in vivo, mice were carefully divided into six groups and treated with PBS, GNS-mPEG, and GNS-pHLIP with and without laser irradiation, respectively. PTT tests were performed after 24 h according to the results of the CT/PA

the cell viability decreased from 79% to 15% at pH 6.4, when the concentration of GNS-pHLIP increased from 0.5 to 4 mg/ L. However, there were no remarkable pH response effects in the GNS-mPEG-treated groups at pH 7.4 and 6.4 (Figure 5b). This finding clearly indicates that the pHLIP-modified GNSs possessed a specific pH response effect and further enhanced the PTT efficiency. Because GNSs with high X-ray absorption have received considerable attention in CT imaging, the potential CT imaging ability of GNS-pHLIP was further evaluated. Tumorbearing mice were intravenously injected with GNS-mPEG and GNS-pHLIP, and then CT images were recorded before and 1, 4, 12, and 24 h after injection. As indicated in Figure 6a, the CT signals of the tumors in both GNS-mPEG- and GNS-pHLIPtreated mice gradually increased, indicating the accumulation of nanomaterials in the tumor. A quantitative analysis of the region of interest drawn around the tumors showed that the CT values of the tumors in the GNS-pHLIP-treated mice reached ∼130 HU at 24 h postinjection, which is significantly higher than that of the GNS-mPEG-injected mice (∼100 HU, p = 0.03) (Figure 6b). PA imaging of the GNS-pHLIP was also examined in vivo. As seen in Figure 6c, the PA images of the tumors were obtained before and 24 h after injection. The PA signal values of the GNS-pHLIP-treated group were significantly higher than those of the GNS-mPEG-treated group (p = 0.04, Figure 6d). This observation was consistent with the results of the CT imaging and ICP assay in vivo. These results F

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Figure 8. (a) Tumor growth curves of tumor-bearing mice after treatment. The tumor volumes were normalized to their initial sizes (asterisks indicate p < 0.05). (b) The changes of body weight in each group after treatment. (c) Staining of the main organs from each group of mice after laser irradiation 15 d postinjection. Scale bars: 100 μm.

4. CONCLUSIONS In this work, we innovatively used a pH-dependent transmembrane activity peptide (pHLIP) to decorate GNSs and successfully designed a pH-response multifunctional agent that could be used for CT/PA-guided photothermal therapy of solid tumors. The introduction of the pHLIPs to the GNSs not only increased the cell uptake of this agent at pH 6.4 but also improved its tumor accumulation because of the acidresponsive ability of the peptide. This effect greatly endowed more effective CT/PA imaging of MCF-7 tumors and a greater PTT effect without additional cytotoxicity. This study provides an important approach that thoroughly takes advantage of the characteristics of the tumor microenvironment and GNSs to achieve tumor-targeting imaging and therapy, which could be valuable for the design of novel multifunctional agents in tumor therapy.

imaging. Tumor sizes and body weights were observed at 15 d after treatment. Figure 8a showed that the tumor volumes in the mice treated with PBS (with and without laser irradiation), GNS-mPEG, or GNS-pHLIP (without laser irradiation) grew rapidly. As compared to the GNS-mPEG-treated group irradiated with the laser, mice injected with GNS-pHLIP after PTT testing showed significant tumor suppression (p = 0.04). Therefore, the GNS-pHLIP exhibited a superior PTT effect in vivo. Furthermore, none of the mice showed apparent weight loss during the observation period (Figure 8b). In addition, H&E staining of the vital organs from each group was carried out and did not reveal an apparent injury, except for the tumors. Tumor pathology staining showed obvious necrosis after the PTT tests in the nanomaterial injection groups, particularly in the GNS-pHLIP treatment group (Figure 8c). These results suggest that the GNS-pHLIP exhibited good biocompatibility, low toxicity, and high PTT efficiency and may be suitable imaging-treatment agents for clinical applications.

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Additional figures showing hydrodynamic diameters of GNS-pHLIP at different pH values, TEM images of GNS-pHLIP before and after irradiation, and optical microscopic images and TEM images of MCF-7 cells incubated with GNS-pHLIP at pH 7.4 and pH 6.4 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Guangming Lu: 0000-0003-4913-2314 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly appreciate financial support from the National Key Basic Research Program of the PRC (2014CB744504), the Major International (Regional) Joint Research Program of China (81120108013), the National Natural Science Foundation of China (81530054, 81672102, 81601556, 81501588, 81501538, 81601555), the Natural Science Foundation of Jiangsu Province (BK20140734), the China Postdoctoral Science Foundation (2016M593035), and Jiangsu Planned Projects for Postdoctoral Research Funds (1501122c).

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DOI: 10.1021/acsami.6b13237 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX