A Gold Nanocage/Cluster Hybrid Structure for Whole‐Body ...to the combined actions of PTT and EGFR...

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A Gold Nanocage/Cluster Hybrid Structure for Whole-Body Multispectral Optoacoustic Tomography Imaging, EGFR Inhibitor Delivery, and Photothermal Therapy

Chenyue Zhan, Yong Huang, Guifang Lin, Shuailing Huang, Fang Zeng,* and Shuizhu Wu*

C. Zhan, Y. Huang, G. Lin, S. Huang, Prof. F. Zeng, Prof. S. Z. WuState Key Laboratory of Luminescent Materials and DevicesCollege of Materials Science and EngineeringSouth China University of TechnologyWushan Road 381, Guangzhou 510640, ChinaE-mail: [email protected]; [email protected]

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smll.201900309.

DOI: 10.1002/smll.201900309

1. Introduction

Gold nanocages (AuNCs) are a class of hollow nanostructures with porous walls. Since their debut over a decade ago,[1] they have gained enormous attention for their potential to achieve various biomedical applications such as bioimaging, and drug delivery and therapy owing to their bioinert nature, tunable localized sur-face plasmon resonance (LSPR) peaks, and high photothermal conversion efficiency.[1–10] In particular, with hollow interiors and porous walls, AuNCs can be readily loaded with various drugs in their interiors with high loading efficacy, making the them ideal for drug delivery in therapeutic applications.

Gold nanocages (AuNCs) and gold nanoclusters (AuClusters) are two classes of advantageous nanostructures with special optical properties, and many other attractive properties. Integrating them into one nanosystem may achieve greater and smarter performance. Herein, a hybrid gold nanostructure for fluorescent and optoacoustic tomography imaging, controlled release of drugs, and photothermal therapy (PTT) is demonstrated. For this nanodrug (EA–AB), an epidermal growth factor receptor (EGFR) inhibitor erlotinib (EB) is loaded into AuNCs, which are then capped and functionalized by biocompatible AuCluster@BSA (BSA = bovine serum albumin) conjugates via electrostatic interaction. Upon cell internalization, the lysosomal proteases and low pH cause the release of EB from EA–AB, and also induce fluorescence restoration of the AuCluster for imaging. Irradiation with near-infrared light further promotes the drug release and affords a PTT effect as well. The AuNC-based nanodrug is optoacoustically active, and its biodistribution and metabolic process have been successfully monitored by whole-body and 3D multispectral optoacoustic tomography imaging. Owing to the combined actions of PTT and EGFR pathway blockage, EA–AB exhibits marked tumor inhibition efficacy in vivo.

Bioimaging and Therapy

One prominent advantage of utilizing AuNCs in biological applications is their straightforward surface modification capa-bility due to their strong interaction with thiolate groups. However, despite the recent advances in modification and func-tionalization for AuNCs,[11–21] the robust surface modification strategies, which can not only enhance the biocompatibility and stability of AuNCs in biological systems, but also efficiently block the pores on AuNCs to prevent premature drug leakage and release their payloads on demand, are highly sought after. Previously, gold nanocluster (AuCluster)–protein conju-gates have been designed and used as a class of biomaterials for drug delivery and bioimaging due to their biocompatible nature and bright red emission.[22–24] In particular, bovine serum albumin (BSA), a widely used biodegradable protein with its isoelectric point at around pH 4.7, has been utilized to reduce gold salts and sta-bilize AuClusters, so as to form highly

biocompatible and stable AuCluster@BSA conjugates.[25,26] In consideration of their advantageous properties, we envision that the AuCluster@BSA conjugates may potentially be used in com-bination with AuNCs to serve as a smart modification agent to cap and stabilize the latter and to control the drug release.

Moreover, with their size being readily tuned within the range from 15 to 200 nm,[6] AuNCs are particularly suitable for drug delivery in cancer therapy, as they can accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect. For tumor treatment, the molecularly targeted anticancer drugs, which have been designed to interfere with the specific biochemical pathway for a specific cancer, have become effective therapeutic drugs for cancer treatment.[27–29] In theory, by targeting and downregulating particular mole-cules involved in the growth and proliferation of cancer cells, the molecularly targeted drugs are more specific to cancer cells than the cytotoxic agents that target cell replication.[30,31] The epidermal growth factor receptor (EGFR) pathway is a pre-ferred target for cancer therapy, as EGFR is overexpressed on many types of solid tumors and plays very important role in facilitating cancer cell proliferation, survival, and invasion.[32] Erlotinib (EB), a FDA-approved tyrosine kinase inhibitor for

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molecularly targeted therapy of several cancers,[33–35] inhibits EGFR signaling pathway and blocks the autophosphorylation of critical tyrosine residues on EGFR.[36] However, being an anticancer drug available as a coated tablet for oral administra-tion, EB faces some drawbacks such as poor oral bioavailability due to its poor solubility and permeability and side effects such as dose-dependent acneiform rash and diarrhea.[37] Previously, several groups including our group have developed some nano-sized carriers for EB delivery to tackle these problems.[38–40] EB is a small-molecule EFGR inhibitor, which could be readily loaded into the AuNC via the 5–8 nm pores with high drug loading due to the large hollow interiors of AuNC.

In addition, the LSPR of AuNCs can be straightforwardly tuned to the near-infrared (NIR) region with high extinction coefficient,[3,19] which endows AuNCs with high photothermal conversion capacity for photothermal therapy (PTT) as well as optoacoustic (photoacoustic) imaging capability. Compared with pure optical imaging (e.g., fluorescent imaging),[41,42] the optoacoustic (OA) imaging technique is an attractive imaging modality that functions by detecting the ultrasound waves gen-erated by the thermoelastic expansion of tissue resulting from the laser pulse absorption.[43–48] This technology is not affected by photon scattering and thus can provide high-resolution optical images deep inside biological tissues.[49–55] Particularly, the multispectral optoacoustic tomography (MSOT),[56,57] which is a spectral optoacoustic technique achieved by irradiating an object with multiple wavelengths and allowing the system to detect ultrasound waves from different photoabsorbing substances in the tissue, has shown its great potential in a wide range of biological imaging. In MSOT imaging, spectral unmixing can be readily exploited to deconvolute the ultra-sound waves from different contrast agents, allowing each of them to be visualized separately. More importantly, 3D imaging can be realized by a volumetric imaging technique, or by ren-dering stacks of 2D images into 3D MSOT images, which could be employed to locate the disease foci.[55] In consideration of these advantages of MSOT technology, using it to track the optoacoustically active drugs in vivo would help to understand their pharmacokinetics upon administration.

In this study, by exploiting the multiple advantages of AuNC, we developed an AuNC-based nanodrug (EA–AB) for MSOT imaging and tumor inhibition via EGFR pathway blockage and photothermal therapy (Scheme 1). This nanodrug was fab-ricated by loading an EGFR inhibitor (EB) into the positively charged AuNC, followed by functionalizing such nanocage with the negatively charged AuCluster@BSA at neutral pH through the strong Coulombic (electrostatic) interaction. In this nano-structure, AuNC serves as the drug carrier and the exogenous contrast agent for MSOT imaging as well as the PTT generator, while the AuCluster@BSA plays multiple roles as the stabi-lizing, capping, and fluorescent imaging agent, and also helps to release EB from EA–AB. By obtaining 3D MSOT images of the test mice, we were able to track the biodistribution of the AuNC-based drug delivery system. Our results also reveal that the nanosystem displays high antitumor efficacy owing to the combined actions of EGFR signaling pathway blockage and photothermal therapy. We suppose the results presented herein may be of great interest for the development of gold-nanostructure-based drug delivery platforms.

2. Results and Discussion

2.1. Fabrication of the EA–AB and Its Drug Release Profile

We performed a series of experiments to fabricate the AuNC-based EA–AB. First, the pristine AuNCs were synthesized via the galvanic replacement reaction with Ag nanocubes as the templates,[1,58] and the AuCluster@BSA conjugates were synthesized separately following a previously reported pro-tocol.[25] Afterward, lipoic acid (LA) was used to replace the orig-inal protecting agents on nanocages, and poly(l-lysine) (PLL, MW = 2000) molecules were employed to modify the nano-cages via amidation reaction with lipoic acid, forming the posi-tively charged gold nanocages (AuNC–LA–PLL). After EB was loaded into AuNC–LA–PLL, the negatively charged AuCluster@BSA conjugates were attached onto the surface of the positively charged gold nanocages by Columbic interaction in neutral pH; eventually, the AuCluster@BSA-modified and EB-containing gold nanocage was attained, which is the final nanodrug and hereinafter referred to as EA–AB. The strong interaction between the oppositely charged polyelectrolytes[59] (polylysine and BSA) under neutral pH is the key to the successful incor-poration of AuCluster@BSA onto AuNC.

The characterizations for EA–AB and relevant structures are presented in Figure 1. The morphologies as revealed by transmission electron microscope (TEM) for Ag nanocube, pristine AuNC, polylysine-modified AuNC (AuNC–LA–PLL), and EA–AB are shown in Figure 1a. The TEM and high-resolution TEM images of AuCluster@BSA are shown in Figure S1a,b (Supporting Information), respectively, and in Figure S1b (Supporting Information) a lattice spacing of 2.36 Å between two (111) adjacent planes in Au is shown, which indi-cates the formation of AuCluster stabilized by BSA. The images in Figure 1a not only demonstrate the successful syn-thesis of these AuNCs, but also show that the conjugation of AuCluster@BSA on AuNCs leads to capping of the pores on the nanocages. The functionalization of nanocages by lipoic acid, PLL, and AuCluster@BSA was also confirmed by zeta potential and hydrodynamic diameter measurement, as shown in Figure 1b,c. After lipoic acid was conjugated onto the surface of the as-prepared AuNC via strong gold–sulfur (AuS) bond, the zeta potential of LA-modified AuNCs (AuNC–LA) decreased from −13.8 to −22.4 mV, while the average hydrodynamic diam-eter remained unchanged (Figure S2, Supporting Information). After PLL was covalently incorporated onto AuNC–LA, the hydrodynamic diameter slightly increased from 60.5 ± 1.6 to 72.3 ± 2.6 nm, and the zeta potential reversed from nega-tive (−22.4 mV) to positive (+19.7 mV). Finally, the negatively charged AuCluster@BSA was conjugated on the surface of AuNC–LA–PLL to form A–AB (the AuCluster@BSA-modified AuNC without loading EB) or EA–AB (the final prodrug), which allowed their hydrodynamic diameters to increase and zeta potentials to reverse again from positive to negative.

The absorption spectra for EB, pristine AuNC, AuCluster@BSA, and EA–AB are presented in Figure 1d. By comparing these spectra, we can find that EA–AB exhibits the character-istic absorption features of all constituent species, namely, the broad LSPR band (from 600 to 900 nm) of pristine AuNC, the narrow absorption band (from 300 to 360 nm) of EB, and the

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AuCluster’s elevating absorption tendency from 300 to 450 nm. This provides additional evidence for the successful drug loading and AuCluster@BSA attachment on the gold nanocages.

Moreover, the covalent incorporation of PLL onto AuNC–LA is further proved by Fourier-transform infrared spectroscopy (FTIR), as shown in Figure 1e. Before incorporation of PLL, AuNC–LA shows strong bands of carbonyl at 1704.69 cm−1 (in COOH) and 1409.18 cm−1 (in COO−).[60] However, after PLL was incorporated onto AuNC–LA via amidation reac-tion, the bands for carbonyl in COOH and COO− were almost replaced by the bands of carbonyl (in amide) at 1672.82 and 1567.05 cm−1, which are from the original amide groups in PLL and the newly formed amide groups as a result of amidation reaction.[60] This result provides further evidence of the forma-tion of AuNC–LA–PLL.

The colloidal stability of pristine AuNCs and the EA–AB was evaluated by recording the hydrodynamic diameters in phos-phate-buffered saline (PBS) and in 100% fetal bovine serum over time, and the result is given in Figure 1f. The stability of EA–AB was further verified by TEM and absorption spectrometry.

As shown in Figures S3 and S4 (Supporting Information), the morphology of dried EA–AB remained unchanged, and only a slight decrease in the absorption at ≈340 nm was observed for EA–AB in PBS over 5 days. It can be seen that, upon modifica-tion by AuCluster@BSA, the EA–AB exhibits much higher col-loidal stability than pristine nanocage as evidenced by the barely changed properties of the EA–AB over 5 days.

SDS-PAGE analysis (Brilliant Blue staining) was performed to further confirm the attachment of AuCluster@BSA on AuNC, and the effect of pH on the interaction of the two com-ponents as well. As revealed in Figure 2a, an intense band was found with a molecular weight less than 70 kDa in the lane of free BSA (lane D). In contrast, no band at this molecular weight in other lanes was observed, indicating the retention of BSA by AuCluster or by the EA–AB. In addition, diffuse bands could be observed at the transition of stacking and separating gel in lane A (for the EA–AB upon incubation in pH 4.5 buffer) and lane C (for AuCluster@BSA in pH 7.4 PBS), which sug-gests that the strong AuSH interaction between AuCluster and BSA prevents the migration of BSA into the separation gel

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Scheme 1. Schematic illustration for a) fabrication of EA–AB and b) its imaging capability and dual actions on tumor cell.

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even at acidic pH (lane C), and some AuCluster@BSA conju-gates could leave the EA–AB at acidic pH environment (lane A). This result also indicates that at neutral pH, the EA–AB is quite stable (as shown in lane B). BSA is made up of zwitterionic peptides, and the net charge of BSA can be positive or nega-tive depending on the environmental pH. The zeta potential for AuCluster@BSA was measured under various pHs in buffers and the result revealed that the isoelectric point of AuCluster@BSA was approximately pH 4.7 (Figure S5, Supporting Infor-mation). This suggests that at pH 7.4, AuCluster@BSA is posi-tively charged and can strongly bind to the negatively charged AuNC, while with the decreasing environmental pH, the zeta potential for AuCluster@BSA increased.

The morphology of the EA–AB after treatment with pH 4.5 buffer solution was observed on a transmission electron micro-scope, and the TEM image (Figure S6, Supporting Information) reveals that the morphology of treated EA–AB could not fully turn back to that of the original AuNC, and the treated EA–AB was still covered with some BSA. In addition, we also measured the hydrodynamic diameters of EA–AB at pH 7.4, pH 6.0, and pH 4.5 using the dynamic light scattering method. As shown in Figure S7 (Supporting Information), the hydrodynamic size of EA–AB increased with the decreasing pH, indicating that at lower pH the interaction between macromolecular chains of BSA and the positively charged nanocage became weak and BSA chains assumed loosely packed state (extended state) on nanocage. In aqueous environments, there are two pairs of interactions governing the chain conformation of BSA: one

is the electrostatic (Coulombic) interaction between BSA and the positively charged nanocage, and the other is the solva-tion interaction between BSA and water molecules. At neutral pH, the electrostatic interaction prevails and BSA can strongly attach to the nanocage, forming a close-packed layer on nanocage surface and hence blocking the drug leakage. With the decreasing pH, the electrostatic interaction gradually weak-ened, and the solvation may eventually become comparable to the electrostatic interaction even before the charge reversal of BSA. As a result, BSA macromolecular chains assumed extended conformation as pH decreased and could not effi-ciently block the small-molecule drug EB from migrating out of the nanocage.

Previous studies have shown the fluorogenic and bio-imaging ability of AuCluster@BSA.[61–64] Interestingly, we found that for EA–AB, the fluorescence of AuCluster@BSA at 650 nm could be quenched at neutral pH via energy transfer process by AuNCs due to the LSPR effect of the latter, as shown in Figure S8 (Supporting Information), while the decrease in environmental pH caused gradual recovery of AuCluster@BSA’s fluorescence (Figure 2b,c). At lower pH, the extended molecular state of BSA induced the fluores-cence recovery of the AuClusters, since at extended state the distance between AuCluster and AuNC became longer and the AuNC could no longer quench AuCluster’s emission via energy transfer process. In contrast, for the AuCluster@BSA without being attached onto AuNC, the change in fluorescent emission with the decreasing pH was far less prominent, as

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Figure 1. Characterization of AuNC-based nanostructures. a) TEM images of I) Ag nanocube, II) AuNC, III) AuNC–LA–PLL, and IV) EA–AB. Scale bar: 20 nm. b) Zeta potential of AuNC, AuNC–LA, AuNC–LA–PLL, A–AB (AuNC–AuCluster@BSA without loading EB), and EA–AB. Columns represent mean ± standard deviation (SD), n = 3. c) Diameter distribution of AuNC, AuNC–LA–PLL, A–AB, and EA–AB as determined by dynamic light scattering. d) UV–vis absorption spectra for EB (15 µg mL−1), AuNC (100 µg mL−1), AuCluster@BSA (50 µg mL−1), and EA–AB (100 µg mL−1) in pH 7.4 PBS solu-tion (dispersion) at 25 °C. e) FTIR spectra of AuNC–LA and AuNC–LA–PLL. f) Evolution of average diameter as determined by dynamic light scattering for EA–AB and pristine AuNC in PBS or serum in 5 days. Data represent mean ± SD, n = 3.

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shown in Figure 2c and Figure S9 (Supporting Information). This emission behavior of EA–AB may allow us to monitor the release of EB by observing the fluorescence enhancement in acidic pHs.

Moreover, the LSPR absorption of AuNCs in the NIR region allows them to serve as excellent photothermal agents.[65–68] To evaluate the photothermal capacity of EA–AB, we irradiated its PBS dispersion (with the concentration of 25 µg mL−1 based on AuNC weight) for 5 min with an 808 nm NIR laser, and the temperatures for the dispersion were recorded and imaged by using an NIR thermal camera. A pure pH 7.4 PBS solution was used as a control. As shown in Figure 2d, the temperate for EA–AB dispersion increased greatly with the increasing irradiation time or laser power density. In contrast, the pure PBS solution only exhibited negligible temperature elevation upon laser irradiation. To confirm the photothermal capability of EA–AB was contributed by AuNC, the temperature eleva-tion curves of AuNC and AuCluster@BSA were determined by irradiating them with 1.5 W cm−2 laser for 5 min. As shown in Figure S10 (Supporting Information), only AuCluster@BSA could not cause significant temperature increase. Clearly, it was the AuNC that contributed to the photothermal conversion. The NIR thermal images given in Figure 2e provide visible evidence for the temperature elevation. Furthermore, the photothermal conversion efficiency of EA–AB has been determined as 85.5% (Supporting Information). This high photothermal conversion efficiency can afford a good foundation for subsequent photo-thermal therapy experiments.

EB is a tyrosine kinase inhibitor and used in cancer therapies.[33–35,38] In this study, EB was loaded into AuNC–LA–PLL nanocage, and the encapsulation efficiencies were determined for EB solutions of varied concentrations. As shown in Figure S11 (Supporting Information), the encapsu-lation efficiency reached a peak value of 24.7% at the EB con-centration of 150 µg mL−1. In the subsequent experiments, we chose 150 µg mL−1 as the test concentration, at which the final drug loading was determined as 52 µg per mg AuNC in EA–AB by UV–vis absorption spectrometry using a prede-termined standard curve shown in Figure S12 (Supporting Information). The in vitro drug release profile was determined in buffer dispersion at varied pH values in a period of 120 min by recording the absorbance of EB at 340 nm. As illustrated in Figure 2f, at neutral pH only small amount of EB was released from EA–AB within 120 min, which suggests the satisfactory capping efficiency and stability of EA–AB under simulated neutral pH physiological condition. Reasonably, our nanodrug is potentially able to prevent any premature leakage, and this can significantly improve the therapeutic efficacy and reduce adverse side effects of drugs. However, at pH 4.5, substantial release of EB was detected. It can also be found in this figure that another significant increase of EB release was observed upon NIR irradiation (1.5 W cm−2, 808 nm), which suggests that higher temperature further enhances the drug release. At higher temperature, the thermal motion of molecules and par-ticles was intensified and they had higher energy to overcome the energy barrier for destruction of Coulombic interactions.

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Figure 2. a) SDS-PAGE analysis (Coomassie Brilliant Blue staining) for EA–AB in A) pH 4.5 sodium acetate buffer and B) pH 7.4 PBS, C) the AuCluster@BSA in pH 7.4 PBS, and D) the pure BSA (62 kDa) in pH 7.4 PBS. b) Fluorescence spectra of AuCluster@BSA in EA–AB in sodium acetate buffer and PBS dispersions (100 µg mL−1 based on AuNC weight) of varied pH values. λex = 410 nm. c) Fluorescence imaging for AuCluster@BSA in EA–AB and pure AuCluster@BSA (50 µg mL−1 in sodium acetate buffer and PBS; the amount of EA–AB is based on AuCluster@BSA weight) at varied pH values (excitation filter: 410 nm; emission filter: 670 nm). The imaging was performed in triplicate. d) Temperature elevation curve for PBS (the control) and EA–AB upon 5 min of laser irradiation with different light intensities. The density of laser power applied to the PBS solution was 1.5 W cm−2. e) Infrared thermal images of PBS (control) and EA–AB dispersion. f) In vitro release profiles of EB from EA–AB at pH 7.4 and pH 4.5. The release profile was determined by recording EB’s absorbance at 340 nm.

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2.2. Cell Imaging and Viability Assays

The cellular internalization of the EA–AB was investigated using 4T1 (a murine mammary carcinoma cell line) cells. As shown in Figure 3a, weak intracellular fluorescence could be observed in 4T1 cells incubated with EA–ABs for 1 h, and after 4 h of incubation, much stronger fluorescence was observed in cytoplasm. Similarly, cell imaging was also performed using L929 cells; as shown in Figure S13 (Supporting Information), relatively weaker fluorescence can also be observed upon 1 and 4 h incubation with EA–AB. This demonstrates that EA–AB

can be readily internalized by the cells and generate intracel-lular fluorescence. Considering there are plenty of proteinases in lysosomes and the biodegradable nature of BSA, we think the lysosomal proteases played major role in the intracellular fluorescence enhancement. We assumed that upon inter-nalization, EA–ABs were transported into lysosomes where proteinases are abundant and pH is low, which is enough to make the BSA chains loosely packed and highly degraded. To verify the location of EA–ABs in lysosome after cellular inter-nalization, we performed cell imaging experiments by labeling lysosomes with LysoTracker Green and staining the nucleus

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Figure 3. a) Fluorescent microscopic imaging for 4T1 cells upon incubation with 25 µg mL−1 (based on AuNC weight) of EA–AB for 1 and 4 h at 37 °C. b) Fluorescent microscopic imaging for 4T1 cells upon incubation with 25 µg mL−1 of EA–AB, 5 µg mL−1 of DAPI, and LysoTracker Green for 1 and 4 h at 37 °C (DAPI was used for nuclei staining). Scale bar: 10 µm. c) Cell viabilities for 4T1 and L929 cells upon treatment with EA–AB or E–AB for 24 h with or without laser irradiation. The concentrations for A–AB and EA–AB are presented based on the weight of AuNC. Data represent mean ± SD from three independent experiments, each performed in eight replicates. d) Representative flow cytometry profiles (annexin V-FITC/PI staining) for 4T1 cells upon varied treatments. 4T1 cells were grouped as PBS, PBS + laser, A–AB (AuNC content: 100 µg mL−1), EB (5.1 µg mL−1), A–AB (AuNC content: 100 µg mL−1) + laser, and EA–AB (AuNC content: 100 µg mL−1) + laser and incubated for 8 h. Laser irradiation was performed at 4 h after drug treatment. Region UL: damaged cells (PI-positive/annexin V-negative); region UR: late apoptotic and dead cells (PI-positive/annexin V-positive); region LR: early apoptotic cells (PI-negative/annexin V-positive); and region LL: vital cells (PI-negative/annexin V-negative).

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with 4′,6-diamidino-2-phenylindole (DAPI) (blue). As shown in Figure 3b, the merged images for the cells after 4 h of incu-bation with EA–AB reveal substantial overlap between the green fluorescence (from the lysosome-specific dye) and red fluorescence (from AuCluster@BSA), which clearly indi-cates the selective accumulation of EA–AB in lysosome. On the other hand, after longer time (6 and 8 h) of incubation, the red and green fluorescence no longer overlapped perfectly (Figure S14, Supporting Information), indicating the migra-tion of AuCluster@BSA (and perhaps EB and AuNC) into cyto-plasm from lysosomes.

To evaluate the effect of EA–AB on cell viability, we per-formed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays for 4T1 cell (an EGFR overexpressing cancer cell line[69]) and L929 cell (an EGFR non-overexpressing cell line) exposed to EA–AB, and the result is shown in Figure 3c. We also performed the MTT assays for 4T1 cells and L929 cells treated with some controls (AuCluster@BSA, the free EB, and the E–AB, an AuCluster@BSA-modified AuNC without loading EB), as shown in Figure S15 (Supporting Infor-mation). As depicted in Figure 3c, incubating 4T1 cells with EA–AB without NIR irradiation decreased viability of 4T1 cells in a dose-dependent manner. On the other hand, exposure to AuCluster@BSA or AuNC resulted in higher cell viability (≈70% or above), while incubation with free EB caused lower cell viability (≈55% or above), as shown in Figure S15 (Sup-porting Information). Figure 3c also shows that, as compared to those tested in the dark, irradiating 4T1 cells with 808 nm laser further decreased viabilities, especially at higher drug dos-ages. In contrast, for L929 cells in which EGFR is not overex-pressed, exposure to EA–AB only slight decreased the viabilities, and further NIR irradiation did not lead to significant reduc-tion in viabilities. Moreover, treatment with other controls (AuCluster@BSA, AuNC, or EB) could not significantly reduce the viability of L929 cells (Figure S15, Supporting Informa-tion). EB also could not remarkably reduce the viability because L929 cells do not overexpress EGFR. These results clearly indi-cate that the controlled release of EB and the PTT effect of AuNC account for enhanced cytotoxicity toward EGFR overexpressing 4T1 cells. Additionally, the cytotoxicity of various formulations toward 4T1 cells was investigated using flow cytometry assay (annexin V-FITC/PI costaining). As shown in Figure 3d, the EA–AB + laser group exhibited highest percentage (78.7%) of apoptosis, which is higher than those of the A–AB + laser group (61.7%) and free EB group (43.5%), and much higher than those of the other groups. This result further verifies the high toxicity of EB–AB toward EGFR overexpressing 4T1 cells.

2.3. In Vivo Distribution of AuCluster Tracked by Fluorescent Imaging and AuNC Tracked by MSOT Imaging

As mentioned earlier, the AuCluster’s fluorescence restored as the EA–AB was exposed to proteinases and/or low-pH environ-ment, and the AuNC exhibited strong optoacoustic signals at pulsed laser irradiation with or without AuCluster@BSA on its surface. Thus, we can use fluorescent imaging to track the AuCluster and use MSOT to track the biodistribution of AuNC, since the two imaging modalities (fluorescence and MSOT)

detected different substances, and the two kinds of signals reflect the different events in vivo and they do not necessarily overlap in the tumor-bearing mice.

We first tracked the biodistribution of AuCluster in the 4T1 tumor-bearing Balb/c nude mice by using fluorescence imaging. Before imaging, the tumor-bearing mice were subject to i.v. injection of the EA–AB (at the dose of 80 mg kg−1, based on AuNC weight), and the mice were sacrificed at 1, 2, 4, 8, and 16 h postinjection. The major organs (heart, liver, spleen, lung, and kidney) and tumor were collected and imaged on a small animal imaging system, and the results are presented in Figure 4a (which shows the fluorescence images) and in Figure S16 (Supporting Information) (which shows the quanti-fied fluorescence intensities). At the first 1 h, the fluorescence in tumor began to emerge, and increased gradually till reaching its highest level at about 8 h, and then disappeared at 16 h as the result of metabolic process. In contrast, the fluorescence in the major organs is not so prominent, and weaker fluores-cence could be observed in the two metabolic organs (liver and kidney) after 4 h upon injection of EA–AB. In lung and spleen, the EA–AB was stable and nonfluorescent since the fluorescence of AuCluster on EA–AB was quenched by AuNC core. As EA–AB accumulated in the tumor tissue owing to the EPR effect, the acidic microenvironment in tumor tissue induced fluorescence restoration of the AuCluster. In addition, the fluorescence images for excised organs and tumors from the sacrificed mice at 8 h after treatment with other controls (A–AB, AuCluster@BSA, or EB) are presented in Figure S17 (Supporting Information), which shows that the treatment with A–AB could induce strong fluorescence in tumor and weaker fluorescence in other organs. This result is consistent with that depicted in Figure 4a. However, treatment with the intrinsically fluorescent AuCluster@BSA resulted in weak fluorescence in all test samples. The size of AuCluster@BSA is small (less than 20 nm); it could neither substantially accumulate in tumor tissue, nor accumulate in other organs due to the fast metabo-lism by the mice. On the other hand, treatment with the non-fluorescent EB produced no fluorescence in all samples.

To achieve noninvasive and real-time tracking of EA–AB, we employed MSOT to image the AuNC in tumor-bearing mice. First, to evaluate the OA efficiency of the EA–AB, we performed phantom imaging experiments by recording optoacoustic (pho-toacoustic) signals from EA–AB solutions of varied concentra-tions. As revealed in Figure 4b, the EA–AB solutions displayed detectable OA signals upon excitation with 805 nm pulsed laser and the intensity increases linearly with the concentration, and this result ensures the subsequent in vivo imaging. The MSOT instrument we used in this study is capable of recording mul-tiple 2D tomographic images and rendering them as orthogonal maximal intensity projection (MIP) images, which can provide 3D information for a specific contrast agent of interest in the ani-mals. Moreover, whole-body MSOT imaging can be achieved this way to afford a complete picture of biodistribution of a contrast agent. On the other hand, we obtained 3D orthogonal whole-body (not including head, neck, and tail) MSOT images through z-stack rendering, and the result is given in Figure 4c. In these images, the anatomical information in grayscale is overlaid with the AuNC information in color. It can be seen from figures that the control group does not exhibit any signal (in color) from

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AuNC. In contrast, at 4 h postinjection of EA–AB (80 mg kg−1, based on AuNC weight), distinct signals from the AuNC can be visualized in tumor, liver, spleen, and lungs, which also verify the accumulation of EA–AB in tumor and some organs (especially metabolic organs such as liver and spleen). The biodistribution of a nanodrug is governed by several factors such as size, surface charge, and surface functionalization. The use of natural carriers can avoid reticuloendothelial system uptake. Previous studies on biodistribution of some nanoparticles revealed that the nanopar-

ticles protected by serum albumin (human serum albumin or BSA) exhibited high retention in liver, lung, and spleen.[70,71] In this study, the BSA-protected AuNC also showed relatively high retention in these organs besides in the tumor region. The time course of MSOT imaging was also obtained and the result is pre-sented in Figure 4d. We can find that, after injection of EA–AB, the MSOT signal from the tumor and some organs increased with time and then gradually disappeared due to metabolism via liver and spleen. In particular, the retention of the AuNCs in tumor

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Figure 4. a) Ex vivo fluorescence images of dissected organs and tumors from sacrificed mice postinjection of EA–AB. The mice treated with PBS were used as controls. Excitation filter: 410 nm; emission filter: 670 nm. n = 3 per group. b) Relative OA intensity of EA–AB in different concentrations excited at 805 nm. c) In vivo orthogonal 3D MSOT view of 4T1 tumor-bearing mice before and after tail vein injection of the EA–AB dispersion (AuNC content: 80 mg kg−1). n = 3 per group. The numbers indicate different organs and tumor regions: 1) liver, 2) spleen, 3) tumor, and 4) lung. d) The x–z 2D overview MSOT images of mice acquired by multispectral data analysis before and after intravenous injection of EA–ABs (AuNC content: 80 mg kg−1) via tail vein. n = 3 per group. Scale bar: 5 mm.

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lasted for more than 12 h. The representative cross-sectional MSOT images at liver, spleen, or tumor region of a tumor-bearing mouse treated with EA–AB are presented in Figure 5a, and the quantified mean MSOT intensities for images are given in Figure S18 (Supporting Information). These images and data can pro-vide detailed and local information on the AuNC distribution. In particular, the magnified cross-sectional images at tumor region clearly reveal the bulged tumor at the right side of mouse as well as the relatively strong MSOT signal within tumor tissue, and this observation verifies the accumulation of EA–AB in tumor tissue.

For comparison, the pristine AuNCs were also intravenously injected into tumor-bearing mice. As shown in Figure S19 (Supporting Information), without modification by AuCluster@BSA, many of AuNCs were detained in tail vein. Moreover, the pristine AuNCs were fast metabolized in liver, and hence they were unable to accumulate in tumor regions.

2.4. EGFR Pathway Blockage by Released EB

EGFR pathway blockage is a potential approach to inhibit the growth of many tumors.[33–35,38] EB is an EGFR inhibitor and

functions through specifically targeting and binding the EGFR tyrosine kinase expressed in various cancer cells, thus blocking the formation of phosphotyrosine residues in EGFR pathway and inhibiting the downstream signal cascades that are essen-tial to the growth, proliferation, and migration of cancer cells. In this study, EB could be released from EA–AB in tumor tissue and cells where pH is low. To verify the EB’s EGFR pathway blocking capability, we evaluated the action of different formu-lations on the expression of EGFR and phosphorylated EGFR (p-EGFR) using Western blotting analyses. Eighteen 4T1 tumor-bearing mice were randomly divided into six groups and the six groups were, respectively, denoted as 1) mice only injected with PBS (control group), 2) mice only injected with A–AB (AuCluster@BSA-modified AuNC without EB) (A–AB group), 3) mice injected with PBS and irradiated with the laser (PBS + laser group), 4) mice only injected with molecular EB (EB group), 5) mice injected with A–AB (no EB loading) with laser irradiation (A–AB + laser group), and 6) mice injected with EA–AB with laser irradiation (EA–AB + laser group). After the tumor-bearing mice were treated with different formulations for 72 h, they were humanely sacrificed and the tumors were excised for Western blotting analysis. As shown in Figure 5b,

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Figure 5. a) Cross-sectional MSOT images at liver, spleen, or tumor site for a 4T1 tumor-bearing mouse at varied time upon injection of EA–AB. The images were merged ones with both signals from EA–AB (in color) and the background (grayscale). The background signal at liver region is strong because the liver contains large amount of blood. The label “1” indicates the spinal cord. The region of interest is marked with red curves. During the data acquisition, the mice were lying on their stomach with certain tilts. b) Western blotting analyses illustrating the level of EGFR and p-EGFR in tumor tissues dissected from 4T1 tumor-bearing mice upon treatment with different formulations: PBS, PBS + laser, A–AB, EB, A–AB + laser, and EA–AB + laser.

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the formulations with no EB (PBS, PBS + laser, A–AB, A–AB + laser) expressed both EGFR and p-EGFR. In contrast, treat-ment with the EB-containing formulations (free EB and EA–AB + laser) did not affect the expression of EGFR, but could block the phosphorylation of EGFR and reduce the expression of p-EGFR, which could subsequently inhibit the downstream signaling of EGFR pathway and eventually lead to the apoptosis of the cancer cells. The result also reveals that the inhibition effect for the EA–AB + laser group is better than that for free EB group. We suppose this may be due to the high solubility in bloodstream, the EPR effect, and the enhanced release of drug upon laser irradiation of the EA–AB + laser group.

2.5. Tumor Inhibition by EA–AB through EGFR Pathway Blockage and PTT

We then assessed the photothermal effects of EA–AB in mice. At 12 h postinjection of PBS (the control) or EA–AB, the in vivo photothermal imaging was performed and the results are pre-sented in Figure 6a,b. Upon NIR light irradiation (1.5 W cm−2) at tumor site for 5 min, mice treated with EA–AB showed local-ized heating in the tumor regions, at which the temperature quickly rose by about 17 °C. This is attributed to the accumula-tion of AuNCs in tumor tissue. However, for the control group, 5 min of irradiation only caused mild increase in temperature at tumor region. These results demonstrate the superior heat generating ability of the AuNC-based drug delivery system.

Next, we evaluated the combined therapeutic efficacy of the EA–AB toward the xenograft tumor in the nude mice, and the data are given in Figure 6c–g. Thirty 4T1 tumor-bearing mice were randomly distributed into six groups (five mice per group) and then treated with six different formulations, respectively. For each treatment, the groups were, respec-tively, intravenously administered with PBS (200 µL), free EB (4.08 mg kg−1 in PBS with 1% dimethyl sulfoxide (DMSO)), or various nanosystems (80 mg kg−1, based on the weight of AuNC). For PTT therapy groups, laser irradiation at 808 nm (1.5 W cm−2) was performed for 5 min at 12 h after injection of different formulations. Figure 6c shows the tumor volume evolution for the six test groups over the 21-day period. One can find that the tumor evolved in a similar way for PBS, PBS + laser, and A–AB groups, and the tumor volumes of these groups all displayed a fast and uncontrolled growth trend, which reveals that without encapsulation of EGFR inhibitor EB, the AuCluster@BSA-modified AuNC (A–AB) itself did not exhibit any tumor inhibition efficacy. In comparison, the A–AB + laser group showed substantial tumor inhibition effi-cacy, indicating that the laser irradiation on the AuNCs can produce strong heat to inhibit the fast growth of the tumor. For the group that only received the treatment of free EB, the tumor grew slightly faster than the A–AB + laser group, which may be attributed to the low solubility of EB in blood-stream. Notably, treatment with the EA–AB followed by the laser irradiation resulted in the strongest efficacy on sup-pressing the tumor growth, which validated that the combi-nation of photothermal effect and EGFR pathway blockage could afford efficient tumor inhibition. In the animal survival experiment, we continuously monitored the survival rates of

the six groups. As shown in Figure 6d, the mice of the PBS, PBS + laser, and A–AB groups were found dead around 30 days after the first treatment (at day 0). In contrast, the mice from the EA–AB + laser group could survive 42 days after the first treatment. By reviewing Figure 6c,d, we can find that at day 21, the average tumor volume for the A–AB + laser group was around 500 mm3 and that for the EB group was over 800 mm3, which belong to advanced-stage tumor for xenograft tumor model of nude mice.[72,73] This suggests that the treat-ments for these two groups were not efficacious enough, and the tumors at advanced stage deteriorated quickly and caused high death rate after the therapies were ceased, as revealed in Figure 6d. As for the EA–AB + laser group, the tumor growth was efficiently inhibited, and tumor volumes stayed at around 200 mm3 during the whole therapeutic course; as a result, no death of mouse was observed from day 20 to day 42.

The representative images of the mice of different groups within the therapeutic process are given in Figure 6e, which provides visualized evidence for the high antitumor efficacy of the EA–AB + laser group. Also, typical images of the dissected tumors from these groups after 21 days of treatment are given in Figure 6f, which reflects the same tendency of tumor inhi-bition efficacy of these treatments. Additionally, hematoxylin and eosin (H&E) staining was utilized to assess the therapeutic action of various treatments.

As demonstrated in Figure 6g and Figure S20 (Supporting Information), the EA–AB + laser group exhibited more promi-nent necrosis compared to other groups. These results fur-ther demonstrated strong antitumor efficacy of the EA–AB. We also monitored body weight for the six groups during the therapeutic process, and we found only the EA–AB + laser and A–AB + laser groups exhibited a certain weight loss (within 10%), and the body weights for other groups remained stable (Figure S21, Supporting Information). Moreover, we could not find any obvious pathological abnormalities in the section (H&E staining) of kidney, liver, lung, spleen, and heart of the mice of various groups (Figure S22, Supporting Information), which suggests the biosafety of the treatments.

3. Conclusion

A combination therapy toward tumor inhibition has been achieved through PTT and ERFR pathway blockage by using an AuNC-based nanodrug. EA–AB was capped by biocompat-ible AuCluster@BSA conjugate that could undergo degradation in the presence of proteinase and assume extended molecular chain conformation under low pH and thereby induce the drug release. The degradation and/or change in molecular chain conformation of AuCluster@BSA also generated strong fluorescence that served as the indication of the drug release. Due to its high absorption in the NIR region, the AuNC-based nanodrug is optoacoustically active, and its biodistribution and metabolic process were successfully monitored by the whole-body and 3D MSOT imaging. Notably, thanks to the combined actions of PTT and EGFR pathway blockage, EA–AB exhibited prominent tumor inhibition efficacy in vivo. We suppose this approach may provide insights for designing other AuNC-based drug delivery systems.

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4. Experimental SectionMaterials: Silver trifluoroacetate (CF3COOAg), chloroauric acid

(HAuCl4), ethylene glycol (EG), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), sulfo-(N-hydroxysulfosuccinimide) (sulfo-NHS), LA, ethyl alcohol (HPLC grade), DMSO (HPLC grade), PLL, and polyvinylpyrrolidone (PVP) were purchased from Aladdin Bio-Chem

Technology Co. Ltd. (Shanghai, China). BSA, LysoTracker Green, DAPI, and MTT were purchased from Life Technology. EB was purchased from Selleckchem. RIPA lysis buffer, protease inhibitor cocktail, phenylmethanesulfonyl fluoride (100 × 10−3 m), bicinchoninic acid assay kit, Coomassie Brilliant Blue fast staining reagent, and the reagents for Western blotting were purchased from KeyGen Biotech Co. Ltd. The water used throughout the experiments was the triple-distilled water

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Figure 6. a) Infrared thermal images of 4T1 tumor-bearing mice intravenously injected with 200 µL PBS (the control) or EA–AB dispersion (AuNC content: 80 mg kg−1) and 12 h later subject to 5 min of NIR laser irradiation (808 nm at 1.5 W cm−2). n = 3 per group. b) Temperature elevation curves of tumor regions of 4T1 tumor-bearing mice 12 h postinjection of EA–AB or PBS. Irradiation time: 5 min; NIR laser source: 808 nm, 1.5 W cm−2. c) Tumor volume evolution for six groups of mice (PBS, PBS + laser, A–AB, EB, A–AB + laser, and EA–AB + laser) (n = 5 per group) with time within 21 days of treatment. d) Percent survival curve of six groups of mice (n = 5 per group) postinjection in 42 days. e) Representative photographs of tumor-bearing mice from six groups at day 7, day 14, and day 21. f) Representative photographs of tumor tissues of six groups dissected from the mice 21 days after the first treatment. g) H&E staining of tumor sections from six groups of mice after 21 days of treatment. Columns represent mean ± SD. The P-values (*P < 0.05, **P < 0.01) were determined using two-sided Student’s t-test.

Zhan

高亮

Zhan

高亮

Zhan

高亮

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(further treated by ion-exchange columns and Milli-Q water purification system).

Preparation of AuNCs: AuNCs were prepared using a galvanic replacement between silver nanocubes and chloroauric acid (HAuCl4) following previously reported procedures.[1,53] Briefly, for the synthesis of Ag nanocubes, 10 mL of ethylene glycol was heated to 150 °C under magnetic stirring in oil bath, and 0.12 mL of 3 × 10−3 m NaSH solution (in EG) was quickly added into the heated solution. After 4 min, 1 mL of HCl solution (which was prepared by adding 4 µL of 12 n HCl into 12 mL of EG) was poured into the mixture, followed by the addition 2.5 mL PVP solution (prepared by adding 20 mg PVP into 3 mL EG) 2 min later. After another 2 min, 0.8 mL of 282 × 10−3 m CF3COOAg solution (in EG) was added into the mixture. The mixture was further stirred at 150 °C for 1 h. The resultant dispersion was washed with acetone once and with purified water twice, and then stored in 4 °C for further use. For preparation of AuNCs, adequate amount of the Ag nanocubes was added into 20 mL of purified water. As the mixture was heated to 90 °C, 0.6 × 10−3 m of aqueous solution of HAuCl4 was injected at a rate of 0.2 mL min−1, and absorption spectrometry was used to monitor the progress. The injection was stopped until the appropriate SPR peak was obtained. The mixture was stirred for another 10 min and the resultant mixture was purified and washed and finally dispersed in purified water.

Preparation of AuCluster@BSA: AuCluster@BSA was synthesized following the previous reported procedures.[25] Briefly, 17 mL of 5 × 10−3 m HAuCl4 aqueous solution was added into 5 mL of 50 µg mL−1 BSA solution at 37 °C, followed by the addition of 0.5 mL of 1 m NaOH solution. The mixture was then incubated at 37 °C for 12 h, and was transformed into dialysis bag (cutoff MW: 14 000 Da) to remove extra BSA and small molecules.

Preparation of PLL-Modified AuNC (AuNC–LA–PLL): To a newly prepared AuNC dispersion (100 µg mL−1, 20 mL), 1.44 mg of LA in 5 mL ethanol was added, and the reaction mixture was stirred under nitrogen atmosphere in the dark at 32 °C for 24 h. The resultant dispersion was collected by centrifugation at 10 000 rpm for 20 min and washed three times with purified water to remove excessive LA and afford LA-modified AuNC (AuNC–LA). Then, AuNC–LA was resuspended in purified water and EDC (2.67 mg) and sulfo-NHS (1.61 mg) were then introduced. The mixture was continuously stirred at 32 °C in dark for 30 min. Afterward, 10 mg of PLL dissolved in 2 mL water was added, and the mixture was stirred for 12 h to afford PLL-modified AuNCs. The LA–PLL–AuNCs were collected by centrifugation at 12 000 rpm for 30 min and washed three times with purified water.

Loading EB into AuNC and Determination of Loading Capacity: 1 mg of EB was dissolved in 0.1 mL DMSO and then added into 100 µg mL−1 dispersion of AuNC–LA–PLL (mixture total volume 10 mL). The mixture was continuously stirred under nitrogen atmosphere in the dark at 32 °C for 24 h. After centrifugation, the resultant product (EB@LA–PLL–AuNCs) was redispersed in 10 mL PBS (pH 7.4). During this process, the supernate was collected to determine the total EB content in supernatants by using the absorption method according to a predetermined standard curve. The EB loading capacity was calculated according to the following equation:

( ) =−

×( ) ( )Loading capacity of EB % 100%

EB original EB supernate

EB@AuNC

M M

M

(1)

where MEB(original) is the mass of fed EB, MEB(supernate) is the mass of determined EB in supernate, and MEB@AuNC is the mass of collected EB@LA–PLL–AuNCs.

Formation of EA–AB and Determination of Loading Capacity of AuCluster@BSA: To pH 7.4 PBS dispersion of EB@LA–PLL–AuNCs (10 mL, 100 µg mL−1), 10 mg of AuCluster@BSA was added. The mixture was stirred for 24 h and the final product (EA–AB) was collected by centrifugation at 12 000 rpm and then washed three times with purified water. During this process, all supernates were collected and dried by lyophilization. The loading capacity of AuCluster@BSA in nanodrug was

measured by the mass differential method and calculated according to the following equation:

Loading capacity % 100%AuCluster@BSA original AuCluster@BSA supernate

EA AB

M M

M( ) =−

×( ) ( )−

(2)where MAuCluster@BSA(original) is the mass of fed AuCluster@BSA, MAuCluster@BSA(supernate) is the mass of determined AuCluster@BSA in supernates, and MEA–AB is the mass of collected EA–AB.

In addition, the ratio of EB (or AuCluster@BSA) relative to AuNC can be determined accordingly using the loading capacity values.

SDS-PAGE Gel Electrophoresis: The effect of low pH on AuCluster@BSA was evaluated by using SDS-PAGE. 1 mL of 100 µg mL−1 EA–AB dispersion was pretreated in pH 4.5 buffer for 30 min followed by centrifugation at 10 000 rpm for 10 min for collection of supernate. In a typical SDS-PAGE experiment, the samples were denatured in the sample buffer containing 60 × 10−3 m Tris (pH 6.8), 25% glycerol, 2% SDS, 14.4 × 10−3 m 2-mercaptoethanol, 0.1% bromophenol blue, and boiled water for 5 min, followed by centrifugation at 5000 rpm for 1 min at 4 °C. Stained molecular weight marker was run along with samples. Electrophoresis was performed using a Bio-Rad Mini Protean gel system at a constant voltage of 80 V for 150 min. After electrophoresis, the gel was stained with Coomassie Brilliant Blue dye and was observed in a Bio-Rad gel imaging system.

Tumor-Bearing Mice Model: Balb/c nude mice (4 weeks old) were provided by Guangdong Laboratory Animal Center, and they were kept under specific-pathogen-free condition with free access to standard food and water. All animal experiments were performed in the Laboratory Animal Center of South China University of Agriculture, and the experiments were carried out under the protocols of the Care and Use of Laboratory Animals of the South China University of Agriculture. 200 µL of 4T1 cells (1.2 × 107 cells mL−1 in pH 7.4 PBS) were injected into subcutaneous tissue at the right hind side of the nude mice to develop the tumor.

MSOT Imaging: In vitro and in vivo MSOT imaging was performed on a multispectral optoacoustic tomography scanner (iThera Medical). For phantom imaging, the control (PBS solution) or the test solution was fully filled in a commercial Wilmad NMR tube, respectively, and then fixed on the holder of the instrument. For in vivo imaging of AuNC-based nanodrug, the mice bearing 4T1 tumor were anaesthetized and scanned with MSOT after intravenous injection of PBS, EA–AB, or AuNC dispersion (in pH 7.4 PBS with AuNC concentration of 10 mg mL−1). Whole mouse body (except for head, neck, and tail) was scanned with the step size of 0.3 mm. The following ten excitation wavelengths were selected in the experiments: 680, 690, 705, 730, 745, 760, 770, 780, 790, and 800 nm (background). After data acquisition, MSOT images were reconstructed using a standard backprojection algorithm. Upon generation of cross-sectional MSOT images, the z-stack images were rendered as orthogonal MIP images or volumetric 3D images. The linear spectral unmixing was utilized to separate signals from the AuNC and those from the absorbers in tissue (e.g., hemoglobin). Three mice were tested for each group in in vivo mouse optoacoustic imaging experiments.

Fluorescence Imaging for Animal Organs: The tumor-bearing mice treated with EA–AB for varied time were humanely sacrificed with CO2 and their organs were excised for fluorescence imaging on an AMI small animal fluorescence imaging system (Spectral Instruments Imaging Co.) with excitation filter of 410 nm and emission filter of 670 nm. All imaging parameters were kept constant.

Photothermal Imaging: 200 µL PBS or 80 mg kg−1 (AuNC content) of EA–AB in PBS was, respectively, injected into the 4T1 tumor-bearing mice through tail vein. The mice were irradiated with NIR laser (808 nm, 1.5 W cm−2) for 5 min at 12 h postinjection. A thermographic map for the tumor-bearing mice after irradiation was imaged by a thermal imaging camera.

Tumor Inhibition Tests: Thirty 4T1 tumor-bearing mice were randomly divided into six groups (five mice per group). When tumors grew up to ≈200 mm3, each group received different treatments (PBS,

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PBS + laser, A–AB, EB, A–AB + laser, and EA–AB + laser). The groups were, respectively, intravenously administered with PBS (200 µL), free EB (4.08 mg kg−1 in 200 µL PBS with 1% DMSO), or various nanosystems (80 mg kg−1, based on the weight of AuNC). For PTT therapy groups, laser irradiation at 808 nm (1.5 W cm−2) was performed for 5 min at 12 h after injection of different formulations. All groups were treated every other day for 21 days. Tumor sizes and body weights were recorded every 2 days during the therapy process. Tumor volume was calculated according to the formula (a × b2)/2, where a and b are the long and short diameters of the tumor, respectively.

Animal Survival Test: Thirty 4T1 tumor-bearing mice were randomly divided into six groups (five mice per group). All the groups were treated in the same way as in tumor inhibition tests except that after 21 days of treatment, the observation lasted for 42 days to evaluate the survival rate of the mice.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgementsThis work was supported by NSFC (21788102, 21875069, 51673066, and 21574044) and the Natural Science Foundation of Guangdong Province (2016A030312002).

Conflict of InterestThe authors declare no conflict of interest.

Keywordsepidermal growth factor receptor (EGFR) pathway, gold nanocages, imaging, multispectral optoacoustic tomography (MSOT), photothermal therapy

Received: January 17, 2019Revised: April 25, 2019

Published online:

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A Gold Nanocage/Cluster Hybrid Structure for Whole‐Body ...to the combined actions of PTT and EGFR...

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