Au₃Cu tetrapod nanocrystals: highly efficient and metabolizable multimodality imaging-guided NIR-II photothermal agents

Abstract

Gold and gold-based alloy nanocrystals (NCs) have been widely used in the field of photothermal therapy (PTT). However, the synthesis of multimodal nano-agents for precise and efficient PTT in the second near infrared (NIR-II) region remains a great challenge. Herein, Au₃Cu TPNCs were developed as a powerful platform for multimodal image-guided photothermal therapy with significant therapeutic effects and good metabolizability in the NIR-II region.

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Conceptual insights

Gold and gold-based alloy nanocrystals (NCs) have been widely used in the field of photothermal therapy (PTT). However, the synthesis of multimodal nano-agents for precise and efficient PTT in the second near infrared (NIR-II) region that could be metabolized in vitro after treatment remains a great challenge. Here, Au₃Cu tetrapod nanocrystals (TPNCs) were prepared by a facile seed-mediated growth method. Owing to their maximum absorption peak in the NIR-II region, the Au₃Cu TPNCs exhibited remarkable photostability, ability for deep tissue photothermal therapy and excellent photothermal performance with a high mass extinction coefficient of up to 53.01 L g⁻¹ cm⁻¹ and 75.27% of photothermal conversion efficiency under irradiation with a 1064 nm laser at a laser power density of 0.8 W cm⁻² for 5 min, and could serve as efficient photoacoustic and photothermal agents. After PEG, Cy5, and folic acid (FA) surface modification, the monodisperse Au₃Cu@PEG-Cy5,FA not only showed excellent multispectral photoacoustic tomography (MOST) and fluorescence imaging potential but also could selectively and quickly eradicate tumors. Furthermore, Au₃Cu@PEG-Cy5,FA could achieve renal clearance through degradation in the tumor microenvironment after the process of PTT. This design paves the way to design a multimodal imaging-guided nanoplatform as a precision theranostic agent in the NIR-II region with appropriate metabolizability.

Traditional cancer treatment methods, including surgery, chemotherapy, radiation therapy and immunotherapy, have many disadvantages and detrimental side effects such as painful recoveries, potential damage to healthy tissues and systems, and an increased risk of tumor metastasis. Photothermal therapy (PTT), using light to cause thermal damage, is a revolutionary approach in tumor therapy because of its spatiotemporal controllability, high efficiency and minimal harm to normal tissues.^1–4 However, without using imaging contrast agents, it is very difficult for PTT to achieve its precise therapeutic effects since its accuracy is compromised. In order to solve such problems, multimodal imaging-guided PTT has recently emerged and achieved rapid development in cancer diagnosis and treatment.^5–9 The combination of multiple imaging techniques could provide more comprehensive and accurate information, enabling us to visualize the size and location of tumors, monitor the treatment process, and analyze the treatment efficiency during PTT.^10–13 However, until now, there have been few all-in-one theranostic nanoagents that have the advantages such as excellent stability and biocompatibility of the resultant composites during therapy and metabolizability after therapy.^14–16 Therefore, it is urgent to develop multifunctional all-in-one nanoagents possessing both theranostic capabilities for PTT and metabolizability after treatment.

Recently, various nanomaterials with good NIR optical properties have been synthesized, many of which have shown favorable photothermal properties in vitro and in vivo. Among these nanomaterials, Au and Au-based nanocrystals (NCs) have attracted much attention in the biomedical field because of their satisfactory biocompatibility, tunable optical absorption/scattering properties for photothermal treatment,^17–23 and easy modification with different moieties (e.g., peptides, DNAs, and antibodies). These nanostructures include nanoshells,^24–26 nanorods,²⁷ nanocages,²⁸ nanostars,²⁹ and Au-based hybrid nanoparticles (NPs).^30–34 The biological transparency windows of the above Au-based photosensitive agents are mainly located in the 650–950 nm range (NIR-I window). The latest research results demonstrate that the second region (NIR-II window) located in the 1000 and 1350 nm range can provide more efficient tissue penetration relative to the NIR-I window.³⁵ In addition, nanoparticles in the size range of 10–30 nm can penetrate deep into the tumor and avoid blood clearance, which is most suitable for tumor targeting therapy.^36–38 For clinical purposes, these nanomaterials must be biodegradable to avoid long-term toxicity.³⁹ However, the synthesis of small sized and metabolizable Au and Au-based NCs with efficient photothermal performance in the NIR-II window still faces challenges.

Herein, Au₃Cu tetrapod nanocrystals (TPNCs) were prepared by a facile seed-mediated growth method for highly efficient multimodal imaging-guided PTT for tumors in the NIR-II window. In the synthesis of Au₃Cu TPNCs, we used Cl⁻ as a surface-confining agent to ensure the growth of flake branches with (111) as the main exposed surface. The length of the arms can be easily adjusted from a few to tens of nanometers by controlling the reaction time. In our current work, we mainly focused on Au₃Cu tetrapods with 22.8 ± 4.6 nm of arm length. Owing to their maximum absorption peak completely located in the NIR-II region, the Au₃Cu TPNCs showed good photostability, deep tissue photothermal therapy, and excellent NIR-II photothermal performance with a large extinction coefficient of 53.0 L g⁻¹ cm⁻¹ and a significantly high photothermal conversion efficiency of 75.27%. PEG, Cy5, and FA modification can respectively endow the NCs with excellent physiological stability, fluorescence imaging capability, and active targeting capacity for cancer cells with high expression of folate receptors. In vivo experiment results demonstrated that intravenous-injected Au₃Cu@PEG-FA can actively target tumor sites through MSOT imaging and eliminate tumors under laser irradiation at 1064 nm. What's more, the Au₃Cu TPNCs could achieve renal clearance through degradation in the tumor microenvironment after the process of PTT. Thus, Au₃Cu TPNCs showed great potential as highly efficient and metabolizable multimodality imaging-guided NIR-II photothermal agents.

The synthesis of Au₃Cu TPNCs was based on a seeded growth approach. Briefly, 3 nm Au seeds were first synthesized according to a previous work (Fig. S1, ESI†).⁴⁰ Then Au seeds were added into an aqueous solution containing HAuCl₄·3H₂O, CuCl₂·2H₂O, D-(+)-glucose, 1-hexadecylamine, and NH₄Cl, which was subsequently heated to 100 °C and kept for 40 min under a nitrogen atmosphere. Because surfactant 1-hexadecylamine could change the polarity of water solution, Au seeds could be dispersed in water solutions under constant stirring conditions. Transmission electronic microscopy (TEM) images revealed that the produced Au₃Cu TPNCs have an average arm length of around 22.8 ± 4.6 nm (Fig. 1A and Fig. S2, ESI†). The high-resolution TEM (HRTEM) image indicated the crystalline structure of the Au₃Cu TPNCs, and a lattice spacing of 0.236 nm corresponded to the (111) planes (Fig. 1B, inset). The selected-area electron diffraction (SAED) pattern (Fig. 1C) proved the preserved bcc structure of the tetrapod because only one set of diffraction spots could be observed. The elemental mapping images in Fig. 1D–F clearly indicated that Au and Cu were homogeneously distributed, which was further confirmed by the EDX spectrum in Fig. S3 (ESI†). A line-scan EDX evaluation (Fig. 1G) further indicated that the two elements present a uniform distribution. The X-ray diffraction (XRD) pattern showed the successful synthesis of Au₃Cu phases (PDF#34-1302) (Fig. 1H), which was consistent with the inductively coupled plasma atomic emission spectroscopy (ICP-AES) result with a Au/Cu molar ratio of about 3 : 1 (Table S1, ESI†). The surface chemical composition of the Au₃Cu TPNCs was analyzed by X-ray photoelectron spectroscopy (XPS). Fig. S4 (ESI†) showed that the Cu, Au, C and N elements coexisted in the NCs. Two weak peaks at 931.6 eV and 951.1 eV were assigned to the Cu 2p_3/2 and Cu 2p_1/2 from Cu⁰ (Fig. S4B, ESI†). The peaks of Au 4f_5/2 at 87.6 and Au 4f_7/2 at 84.1 eV were attributed to the Au atoms with a chemical valence of zero (Fig. S4C, ESI†).

Fig. 1 (A) TEM images of Au₃Cu TPNCs. Inset: The proposed geometric model of Au₃Cu TPNCs. (B) HR-TEM images of Au₃Cu TPNCs. (C) The selective area electron diffraction (SAED) pattern of Au₃Cu TPNCs. (D–F) EDS-Mapping of Au₃Cu TPNCs. (G) EDS line scan of Au₃Cu TPNCs. (H) XRD patterns of Au₃Cu TPNCs. Inset: Unit cell of Au₃Cu TPNCs. (I) UV-Vis-NIR extinction spectra of Au₃Cu TPNCs.

From the extinction spectra, the Au₃Cu TPNCs exhibited a weak extinction coefficient at ∼510 nm, a strong broad peak in the spectral region of 700–1400 nm, and full coverage of the desired biological windows I (650–950 nm) and II (1000–1350 nm) (Fig. 1I). The weak peak at ∼510 nm corresponded to the transverse plasmon absorption, and the strong and broad resonance in the range of 700–1400 nm originated from the longitudinal plasmon resonance, which was explained by the high aspect ratio of the branches.^41,42 The wide range of NIR extinction coefficients of Au₃Cu TPNCs made them an outstanding candidate for PTT.

The growth and evolution processes of the Au₃Cu TPNCs in our system were monitored by TEM at different time intervals. As shown in Fig. 2 and Fig. S5 (ESI†), when the reaction was carried out for 5 minutes, the NCs began to deform, and a few NCs began to grow with branches. When the reaction was kept for 40 minutes, the tetrapod morphology progressively became more evident with the enlargement of size. Subsequently, when the NCs grew up to one hour, only the arm length of the tetrapod NCs grew further. At the same time, we also used UV-Vis-NIR absorption spectroscopy to trace the growth process of the nanostructures. The experiment results showed that the maximum absorption peak of the nanostructures was shifted from 510 nm to 1265 nm (Fig. S6, ESI†). These results indicated that the branched feature of the Au₃Cu TPNCs was not caused by etching, although etching is an effective way to create branched noble metal nanostructures.^43,44 During the reaction, D-(+)-glucose acted as a reducing agent for two metal salts (hydrogen tetrachloroaurate(III) hydrate and copper(II) chloride dehydrate). There was excessive glucose in the reaction system. The kinetic process of both reactions was very similar. Moreover, a certain amount of Cl⁻ ions played a significant role in the formation of the tetrapod structure because no Au₃Cu TPNCs were obtained when Cl⁻ ions were replaced with other halide ions (F⁻, Br⁻ and I⁻) (Fig. S7, ESI†). There is still ongoing work aimed at revealing the mechanism of how Cl⁻ ions influence the phase and shape of nanostructures although it is widely accepted that the presence of Cl⁻ ions can affect the surface energy of nuclei.^45,46 TEM images of the obtained NCs with different n_Au/n_Cu ratios are provided in Fig. S8 (ESI†). These results demonstrated that the n_Au/n_Cu ratio can influence the final shape of the nanoparticles.

Fig. 2 (A) Schematic diagram of the NC growth process. TEM images of the Au₃Cu TPNCs at different growth stages: (B) 0 min, (C) 5 min, and (D) 40 min.

To endow them with excellent physiological stability, fluorescence imaging capability, and specific targeting capacity for cancer therapy, hydrophobic Au₃Cu TPNCs were further modified with HS-PEG-NH₂, HS-PEGNH-Cy5, and HS-PEGNH-FA, respectively (Fig. S9 and S10, ESI†). The successful conjugation of Cy5 and FA onto the surface of the Au₃Cu TPNCs was demonstrated by FT-IR spectra (Fig. S11, ESI†), UV-Vis absorption spectra (Fig. S12, ESI†) and fluorescence spectrum (Fig. S13, ESI†). Through calculations we also found that there were about 486 and 430 FA molecules per Au₃Cu@PEG-FA and Au₃Cu@PEG-Cy5,FA, respectively (Fig. S14, ESI†). TEM images showed that the morphology, size, and crystal lattice of the Au₃Cu TPNCs have no change after this modification (Fig. S15, ESI†). The corresponding hydrodynamic diameter of the Au₃Cu TPNCs increased from 39.87 nm to 44.93 nm after the formation of Au₃Cu@PEG-Cy5,FA (Fig. S16, ESI†), and the zeta potential achieved −3.02 mV (Fig. S17 and Table S2, ESI†), which was suitable for biomedical application. The resultant Au₃Cu@PEG-Cy5,FA displayed excellent stability in PBS solution, deionized water, and RPMI 1640 cell culture medium with 10% FBS, respectively (Fig. S18 and Table S3, ESI†). It was found that they could remain stable for at least 15 days in PBS solution (Fig. S19, ESI†).

As shown in Fig. S20 (ESI†), a strong NIR-II absorption of Au₃Cu@PEG TPNCs was observed in water. There are two main parameters that determine the photothermal performance of nanomaterials, the photothermal-conversion efficiency (η) and extinction coefficient (ε).⁴⁷ Combining the Lambert–Beer law (ESI†) with UV-Vis-NIR absorbance spectra of Au₃Cu@PEG TPNC dispersions at various concentrations (Fig. S20A, ESI†), we calculated the mass extinction coefficient values of Au₃Cu@PEG TPNCs to be 33.1 L g⁻¹cm⁻¹ at λ = 808 nm (Fig. S20B, ESI†) and 53.0 L g⁻¹ cm⁻¹ at λ = 1064 nm (Fig. S20C, ESI†), which show more obvious advantages compared with previously reported inorganic photothermal agents with small size.^4,5,47 The photothermal effect of Au₃Cu@PEG TPNCs in vitro was evaluated under 808 and 1064 nm irradiation at laser densities of 1.0 W cm⁻² and 0.8 W cm⁻² respectively. As shown in Fig. 3A and B, the temperature of Au₃Cu@PEG TPNC dispersions rapidly increased at the beginning and then reached a plateau after 5 min of laser irradiation both at 880 nm and 1064 nm versus no significant temperature increase in the control sample of water (Fig. S21, ESI†), which demonstrates the superior photothermal property of Au₃Cu@PEG TPNCs. Interestingly, the temperature increase under the 1064 nm irradiation treatment is greater than that at 808 nm (Fig. 3C), which corresponds to the higher absorption strength of Au₃Cu@PEG TPNCs at 1064 nm. Clear power-density-dependent temperature was shown at 808 nm (Fig. 3D) and 1064 nm (Fig. 3E). According to the method of Roper and co-workers's report (detailed in the ESI†), the photothermal-conversion efficiency (η) values of Au₃Cu TPNCs were calculated to be 39.45% at 808 nm and 75.27% at 1064 nm (Fig. 3F and G). The morphology of Au₃Cu@PEG TPNCs revealed no significant change after irradiation (Fig. S22, ESI†). It is worth noting that the photothermal-conversion efficiency value at 1064 nm was higher than those of previously reported articles and other morphologies of gold–copper bimetallic nanostructures (Fig. S23–S25, Tables S4 and S5, ESI†).^1,3 The photothermal effect of the Au₃Cu@PEG TPNCs remained stable after four cycles (Fig. 3H and I).

Fig. 3 Temperature curves of Au₃Cu@PEG TPNC dispersions with concentrations of 0, 10, 20, 40, 60 and 80 μg L⁻¹ under irradiation at (A) 808 nm at a laser power density of 1.0 W cm⁻² and (B) 1064 nm at a laser power density of 0.8 W cm⁻² for 5 min respectively. (C) Plot of temperature change versus Au₃Cu@PEG TPNC concentration after laser exposure for 5 min with the 808 nm laser (black line) under 1.0 W cm⁻² and the 1064 nm laser (red line) under 0.8 W cm⁻² for 5 min respectively. Temperature curves of Au₃Cu@PEG TPNC dispersions with different power densities (0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 W cm⁻²) under (D) 808 nm and (E) 1064 nm laser irradiation. Calculation of the photothermal-conversion efficiency value of Au₃Cu@PEG TPNCs at (F) 808 nm and (G) 1064 nm (black line: photothermal effect of Au₃Cu@PEG TPNCs under laser irradiation for 5 min (laser on) followed by 5 min of natural cooling to room temperature (laser off); blue line: time constant (τ_s) of the system on heat transfer decided by using the linear time data during the cooling period). Temperature curves of Au₃Cu@PEG TPNCs for four laser on/off cycles under (H) the 808 nm laser with 1.0 W cm⁻² and (I) the 1064 nm laser with 0.8 W cm⁻².

In order to illustrate the greater penetration depth of the NIR-II window than the NIR-I window, the residual NIR laser energy changes at different depths of tissues with various laser densities were measured (Fig. S26A and B, ESI†). As shown in Fig. S26C and D (ESI†), compared with the 808 nm NIR-I laser, there is a stronger energy attenuation trend while using the 1064 nm NIR-II laser with increased tissue thickness. Normalized penetration energy was plotted versus tissue depth and numerically fitted by an exponential decay curve, in which attenuation coefficients of 0.9245 and 0.8982 were obtained for 808 nm and 1064 nm respectively (Fig. S26C and D, inset, ESI†). In addition, the tissue penetration performance of Au₃Cu@PEG TPNCs under 808 and 1064 nm laser irradiation was also evaluated. As shown in Fig. 4A and B, when the NIR-I and NIR-II lasers penetrated the same thickness of tissue, the attenuation of photothermal heating of the NIR-II laser was weaker than that of the NIR-I laser (Fig. 4C). Overall, the deep tissue penetration performance of Au₃Cu@PEG TPNCs in the NIR-II region showed great promise for in vivo photothermal therapy.

Fig. 4 Evaluation of deep-tissue penetration performance in the NIR-I and NIR-II windows. (A) Schematic diagram and (B) photothermal conversion demonstration equipment of aqueous suspensions of Au₃Cu@PEG TPNCs by a tissue-penetrating NIR laser (0.8 W cm⁻², 40 μg mL⁻¹). (C) The temperature change of aqueous suspensions of dispersed Au₃Cu@PEG TPNCs upon exposure to tissue-penetrating NIR-I and NIR-II lasers.

To assess the cytotoxicity and PTT efficacy of Au₃Cu@PEG TPNCs, NIH3T3 and KB cells were used. As KB cells overexpress the folate receptor (FR), Au₃Cu@PEG-FA can specifically bind to the surface of KB cells through interactions between FA and overexpressed FR. Fig. 5A shows that cell viability was higher than 90% for both NIH3T3 and KB cells with concentrations of TPNCs up to 40 μg mL⁻¹, indicating the very low cytotoxicity of Au₃Cu@PEG-FA. Subsequently, we examined the enhanced phototherapy efficacy in the case of KB cells treated with Au₃Cu@PEG and Au₃Cu@PEG-FA after exposure to the 1064 nm laser (0.8 W cm⁻²) for 5 min. As shown in Fig. 5B, a more significant laser-induced therapeutic effect was observed for Au₃Cu@PEG-FA. Meanwhile, no cytotoxicity was found in the cases with only Au₃Cu@PEG-FA or only laser irradiation. The laser-triggered PTT effect was further evaluated by live/dead cell staining experiments. As shown in Fig. 5C, green fluorescence was observed in cells treated with only Au₃Cu@PEG-FA or only laser irradiation. However, red fluorescence was observed in cells treated with Au₃Cu@PEG-FA under laser irradiation. The amount of dead cells treated with Au₃Cu@PEG-FA was higher than that with Au₃Cu@PEG under the same conditions, which was consistent with the CCK8 assay results (Fig. 5B). These results demonstrated that Au₃Cu@PEG-FA can specifically kill the FR overexpressed cancer cells by photothermal treatment. In addition, no reactive oxygen species (ROS) were detected in this system (Fig. S27, ESI†), further indicating that such a process was mainly hyperthermia-induced cancer cell death. Additionally, the specific targeting ability of Au₃Cu@PEG-Cy5,FA toward the FR overexpressed cancer cells was also confirmed by fluorescence imaging (Fig. S28, ESI†).

Fig. 5 In vitro photothermal cell ablation. (A) Viabilities of the KB (black) and NIH3T3 (red) cells determined by CCK8 assay after incubation with various concentrations of Au₃Cu@PEG-FA for 24 h. (B) Viabilities of KB cells after incubation with various concentrations of Au₃Cu@PEG TPNCs and Au₃Cu@PEG-FA induced photothermal therapy under 1064 nm 0.8 W cm⁻² laser irradiation. (C) Fluorescence microscopy images of (left) live cells, stained with Calcein-AM; (middle) necrotic or apoptotic cells, stained with PI; (right) merged, incubated with Au₃Cu@PEG-FA, and irradiated with laser; KB cells incubated with Au₃Cu@PEG TPNCs and irradiation; KB cells incubated with Au₃Cu@PEG-FA only; KB cells irradiated with laser only; and KB cells without any treatment, respectively (from top to bottom). In all the laser irradiation experiments, irradiation was at a power density of 0.8 W cm⁻² for 5 min. Error bars, mean ± SD.

Based on the favorable in vitro results, in vivo tumor-targeting ability of Au₃Cu@PEG-FA on mice bearing KB tumors was also evaluated by fluorescence imaging. After Au₃Cu@PEG-Cy5 and Au₃Cu@PEG-Cy5,FA (20 mg kg⁻¹, 200 μL) were injected into nude mice bearing the KB tumor, respectively, fluorescence images were taken. As shown in Fig. 6A, strong fluorescence signals were mainly present in the tumor site after 24 h. For Au₃Cu@PEG-Cy5, however, no fluorescence signal appeared in the tumor site after 24 h. In addition, the targeting capacity of Au₃Cu@PEG-Cy5,FA was assessed by ex vivo imaging of the main organs and tumors of mice at 48 h after injection. As shown in Fig. 6B, although the liver and spleen have strong signals, fluorescence signals are clearly observed in the tumor. Moreover, it is noteworthy that a higher tumor site signal was observed in mice injected with Au₃Cu@PEG-Cy5,FA than with Au₃Cu@PEG-Cy5, which was consistent with the ICP-MS results (Fig. S29, ESI†). These results suggested that Au₃Cu@PEG-Cy5,FA accumulated more at the tumor sites than Au₃Cu@PEG-Cy5, because the former one had active targeting relying on FA and the FA receptor in addition to passive targeting.^49–52 In addition, the Au₃Cu@PEG-Cy5,FA exhibited a longer retention time (48 h) in vivo, which was confirmed by the semi-quantified fluorescence intensity (Fig. 6C).

Fig. 6 (A) Real-time fluorescence images of tumor-bearing mice at different time points after intravenous injection of Au₃Cu@PEG-Cy5 and Au₃Cu@PEG-Cy5,FA into tumor bearing mice. (B) Ex vivo fluorescence images of the major organs (1, liver; 2, spleen; 3, lung; 4, heart; 5, kidney; 6, tumor) harvested at 48 h post-injection. (C) The average fluorescence intensities of the tumor site after intravenous injection of Au₃Cu@PEG-Cy5 and Au₃Cu@PEG-Cy5,FA, respectively. (D) Real-time MSOT images of tumor-bearing mice at different time points after intravenous injection of Au₃Cu@PEG-Cy5 and Au₃Cu@PEG-Cy5,FA into tumor bearing mice. (E) The average MSOT signal intensities of the tumor site after intravenous injection of Au₃Cu@PEG-Cy5 and Au₃Cu@PEG-Cy5,FA, respectively. Error bars, mean ± SD, *P < 0.05 (two-tailed Student's t test).

Next, the in vivo tumor-targeting ability of Au₃Cu@PEG-Cy5,FA in mice bearing KB tumors was evaluated by MSOT imaging. In vivo MSOT imaging is noninvasive and provides easy to collect anatomical and functional information, since it has higher penetration and higher spatial resolution. As shown in Fig. 6D, after the injection of Au₃Cu@PEG-Cy5,FA for 3 h, the photoacoustic signal was detected around the tumor, indicating the ability of Au₃Cu TPNCs as agents for MSOT imaging in vivo. Moreover, the photoacoustic signal intensity in tumors injected with Au₃Cu@PEG-Cy5,FA was higher than that of the mice injected with Au₃Cu@PEG-Cy5, suggesting higher accumulation and deeper penetration of Au₃Cu@PEG-Cy5,FA at the tumor sites, which is consistent with the fluorescence imaging results (Fig. 6A). We further studied the different signals of the tumor region in more detail after injection of Au₃Cu@PEG-Cy5 and Au₃Cu@PEG-Cy5,FA respectively. As shown in Fig. 4E, after injection of Au₃Cu@PEG-Cy5,FA for 24 h, the photoacoustic signal intensity of the tumor was increased by about 380%, while the enhancement was less than 208% for Au₃Cu@PEG-Cy5, indicating higher accumulation and deep penetration of Au₃Cu@PEG-Cy5,FA at the tumor sites.

Biodegradation into nontoxic subparts is an ideal property for injected inorganic nanoparticle based PTT, as long-term toxicity of the carriers would be of less concern.⁴⁸ To evaluate the metabolizability of Au₃Cu TPNCs, we dispersed Au₃Cu@PEG in phosphate buffer at pH = 7.4 and pH = 5.0. As shown in Fig. S30A and B (ESI†), there was no obvious change in morphology and size of Au₃Cu@PEG from the TEM images of Au₃Cu@PEG dispersed in phosphate buffer (pH = 7.4) between 0 and 8 days. Nevertheless, the branch of the Au₃Cu TPNCs could be degraded when the pH value was changed to 5.0, which is close to the pH value of the tumor microenvironment (Fig. S30C and D, ESI†). The transformation of the UV-Vis-NIR absorption spectrum of Au₃Cu@PEG dispersed in phosphate buffer with pH = 5.0 after different times (0, 2, 4 and 8 d) gave a reasonable explanation for the above results (Fig. 7A). The absorption peak in NIR-II showed a big blue shift from 1278 nm (0 d) to 1180 nm (8 d). Furthermore, the ratio A1278 nm/A1278 nm (0 d) was bigger than A1180 nm/A1278 nm (8 d). These results indicated a possibility that the Au₃Cu TPNCs could have degraded into smaller nanoparticles. To further prove the above speculation, the biodegradation behavior and structural evolution of Au₃Cu@PEG were further evaluated in cells (Fig. 7B). After 8 days of intracellular co-incubation, Au₃Cu TPNCs were almost degraded into ultrasmall nanoparticles with the same phase as the initial Au₃Cu TPNCs (Fig. S31, ESI†). The reason for the degradation of Au₃Cu TPNCs was that copper was more active than gold on the nanoscale. The oxidation of copper on the surface of Au₃Cu TPNCs could lead to the instability of the entire nanostructure under acidic conditions. And then they were degraded into smaller nanoparticles. For most of the inorganic nanoparticles renal clearance has been demonstrated to be the most practical means for evading undesirable toxic effects. One of the major challenges facing renal clearable inorganic nanoparticles is their specific accumulation in tumor tissues in vivo. The real-time fluorescence images of tumor-bearing mice at different time points (0, 1, 2, 4, 6 and 8 d) after intravenous injection of Au₃Cu@PEG-FA,Cy5 into KB-tumor bearing mice are shown in Fig. 7C. After 2 days, Au₃Cu@PEG-FA,Cy5 NCs were mainly accumulated in the kidney of mice from the fluorescence images. However, the fluorescence signal became weaker and weaker after 4 days. This result was consistent with the above findings. In addition, we collected the urine of mice and carried out TEM characterization (Fig. 7D and inset). A large number of nanoparticles appeared in the TEM image. To sum up, Au₃Cu@PEG-FA,Cy5 could undergo renal clearance in vivo after degradation in the tumor microenvironment.

Fig. 7 (A) UV-Vis-NIR absorption spectrum of Au₃Cu@PEG dispersed in phosphate buffer with pH = 5.0 after different times (0, 2, 4 and 8 d). (B) Bio-TEM images of KB cells incubated with Au₃Cu@PEG for different times (0, 1, 2, 4, 6 and 8 d). (C) Real-time fluorescence images of tumor-bearing mice at different time points (0, 1, 2, 4, 6 and 8 d) after intravenous injection of Au₃Cu@PEG-FA,Cy5 into KB-tumor bearing mice. (D) TEM image of the urine of mice which was collected after injection of Au₃Cu@PEG-FA,Cy5 into KB-tumor bearing mice.

The PTT capability of Au₃Cu@PEG-Cy5,FA was monitored by using a KB tumor mouse model. Mice bearing 200 mm³ KB tumors were randomly divided into five groups: (1) Au₃Cu@PEG-Cy5,FA and laser irradiation; (2) Au₃Cu@PEG TPNCs and laser irradiation; (3) only Au₃Cu@PEG-Cy5,FA; (4) only saline; (5) saline and laser irradiation. Each group contained five mice. After an intravenous injection of 200 μL of saline or 20 mg kg⁻¹ TPNCs for 24 h, mice were exposed to a 1064 nm laser (0.8 W cm⁻²) for 5 min. As shown in Fig. 8A and B, the local temperature of the tumor site in mice treated with Au₃Cu@PEG-Cy5,FA and laser irradiation rapidly increased from 37 °C to 56.8 °C within 5 min. Hyperthermia relies on the generation of heat at a tumor site to activate degradation mechanisms of cancer cells leading to cell death. When the local temperature of the tumor is higher than 42 °C, the tumor cells would be killed.^53–55 These experiment results suggested that Au₃Cu@PEG-Cy5,FA was sufficient to ablate tumors under laser irradiation. In addition, compared with the other four groups, a significant inhibition and elimination of tumor volume further demonstrated the excellent PTT efficacy of Au₃Cu@PEG-Cy5,FA in vivo (Fig. 8C). As in the other groups, a decrease in the weight of the mice was not observed during the treatment (Fig. 8D), which further demonstrated the low toxicity of the TPNCs. Moreover, the tumor of harvested mice injected with only Au₃Cu@PEG-Cy5,FA was completely eradicated without further reoccurrence after treatment (Fig. 8E).

Fig. 8 (A) Real-time thermal IR images of tumor-bearing mice at different time points after intravenous injection of Au₃Cu@PEG-Cy5 and Au₃Cu@PEG-Cy5,FA into tumor bearing mice under 1064 nm laser irradiation. (B) Temperature change curves of tumor-bearing mice at different time points in the different treatment groups. (C) Volume change of tumor at different time points in the different treatment groups. (D) Body weight change of mice in the different treatment groups. (E) Digital photos of KB-tumor-bearing mice and their tumor regions with different treatments for 15 days. (F) H&E staining for pathological changes in tumor tissues from each group to reveal the effectiveness of in vivo photothermal therapy after different treatments.

A typical hematoxylin and eosin (H&E) method was used to examine the potential toxicity of Au₃Cu@PEG-Cy5,FA in vivo. As indicated in Fig. 8F, there was severe damage to tumor cells of mice as evidenced by cell necrosis and apoptosis in the group of injection with Au₃Cu@PEG-Cy5,FA and laser irradiation. Mice treated with Au₃Cu@PEG-Cy5 and laser irradiation showed less necrotic areas. Negligible necrosis was found in the other three groups. Also, the heating curves of Au₃Cu@PEG-Cy5 and Au₃Cu@PEG-Cy5,FA solutions had no obvious difference under laser irradiation (Fig. S32, ESI†). These results showed that Au₃Cu@PEG-Cy5,FA NCs were efficient as selective targeting nanomaterials with PTT effects in FR overexpressed mice models. In addition, there was no obvious damage and inflammation in the major organs (e.g., spleen, heart, lung, liver, and kidney) of mice that were harvested from the treatment group after H&E staining (Fig. S33, ESI†).

Conclusions

In summary, Au₃Cu TPNCs were synthesized through a facile seed-mediated growth method, which were further demonstrated to serve as multimodality imaging-guided NIR-II agents with metabolizability. Since their maximum absorption peak completely located in the NIR-II region, the Au₃Cu TPNCs exhibited good photostability, ability for deep tissue photothermal therapy, and excellent NIR-II photothermal performance with a large extinction coefficient of 53.0 L g⁻¹ cm⁻¹ and a significantly high photothermal conversion efficiency of 75.27% under 1064 nm laser irradition. In vivo MSOT imaging, flourescence imaging and NIR thermal imaging results indicated that Au₃Cu TPNCs modified with FA exhibited significantly improved tumor active targeting performance. The obtained Au₃Cu@PEG-Cy5,FA NCs could selectively and quickly eradicate tumors in the NIR-II biowindow without obvious side effects in tumor-bearing mice. Furthermore, Au₃Cu@PEG-Cy5,FA could achieve renal clearance through degradation in the tumor microenvironment after the process of PTT. This study opens up a novel approach to develop gold–copper bimetallic nanostructures as a powerful platform for application in multimodal image-guided photothermal therapy with significant therapeutic effects and good metabolizability in the NIR-II region.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The work was supported by the National Natural Science Foundation of China (51672010, 21671088, 21431002, 51631001, 81421004, 51590882, 51602285, 21575161), the National Key R&D Program of China (2017YFA0206301 and 2016YFA0200102), and the Key Laboratory of Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Chinese Academy of Sciences (No. NSKF201607).

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Footnotes

† Electronic supplementary information (ESI) available: See DOI: 10.1039/c8nh00135a

‡ These authors contributed equally to this work.

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