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Tube-Like Gold Sphere–Attapulgite Nanocomposites with a High Photothermal Conversion Ability in the NearInfrared Region for Enhanced Cancer Photothermal Therapy Ping Wu, Dan Deng, Jingwen Gao, and Chenxin Cai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02270 • Publication Date (Web): 07 Apr 2016 Downloaded from http://pubs.acs.org on April 8, 2016

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ACS Applied Materials & Interfaces

Tube-Like Gold Sphere–Attapulgite Nanocomposites with a High Photothermal Conversion Ability in the Near-Infrared Region for Enhanced Cancer Photothermal Therapy

Ping Wu, Dan Deng, Jingwen Gao, and Chenxin Cai* Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, P.R. China.

* Corresponding author, E-mail: [email protected] (C. Cai)

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ABSTRACT: Near-infrared (NIR)-induced photothermal therapy (PTT) is now considered to be a promising and highly efficient method for tumor therapy. Photothermal agents play a crucial role in PTT, and they are required to possess the ability to harvest NIR light and transform the photon energy into heat energy. This work reports a facile method to synthesize a new PTT agent, which is based on the electrostatic binding of the Au nanospheres (Au NSs, ~15 nm) to the surface of a nanometer-sized mineral, attapulgite, to form tube-like Au–attapulgite nanocomposites. These nanocomposites consist of numerous Au NSs, which are linked to each other along the attapulgite surface. The nanocomposites exhibit similar LSPR absorption characteristics to those of Au nanorods with a longitudinal absorption mode that shifts to the NIR region (approximately 670 nm). Moreover, the nanocomposites have a high Cabs/Csca ratio (cross section of absorption to scattering) and photothermal conversion efficiency of 25.6%. Their photothermal therapy effect is studied using A549 cells and A549-cell-bearing nude mice as examples. The results indicate that the nanocomposites can be effectively taken up by the cells, and the nanocomposites show good biocompatibility. The A549 cells almost died after they were incubated with the nanocomposites (at 100 µg mL–1) for 12 h and irradiated by an 808-nm laser with a power density of 0.5 W cm–2 for 15 min. The tumors of nude mice can also be effectively ablated without regrowth during the period of observation (at least 10 days) after photothermal therapy.

KEYWORDS: Gold nanospheres; Au-attapulgite nanocomposites; Attapulgite; Photothermal therapy; Cancer.


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INTRODUCTION With the growing worldwide health problem caused by cancer, the development of a highly efficient therapeutic approach is becoming more urgent than ever before. Chemotherapy, X-ray radiotherapy, and their combination are currently the most commonly used methods for cancer treatment modalities in the clinic. However, these therapeutic approaches have serious undesired side effects, e.g., both normal and cancerous cells are killed indiscriminately.1-5 Photothermal therapy (PTT), which relies on laser-induced hyperthermia to kill cancer cells,6 is a promising method because cells exposed to temperatures of 41‒ 47 ºC will suffer irreversible damage. This damage is due to protein denaturation and cell membrane damage, thus leading to the growth suppression and ablation of tumors.6-8 Moreover, it has been shown that cancerous cells are more prone to be destroyed at an elevated temperature than the healthy ones because of their poor blood supply.8 Compared with traditional methods, PTT exhibits a high selectivity towards cancerous tissues and even cancer cells because this technique can selectively irradiate the tumor tissues and cancer cells by adjusting the position and the irradiation area of the laser to concentrate the laser light on the tumor site. From a practical point of view, the PTT technique also offers several advantages over chemotherapy and X-ray radiotherapy, such as simpler preformation, fewer complications, shorter hospitalization, faster recovery, reduced costs, etc. Although PTT methods have several heating modes,9-13 the NIR laser-induced PTT is an effective and desirable one for biomedical applications because the NIR light (in the region of 650–1400 nm; usually, 650‒950 nm is referred to as the NIR-I window, and 1000‒1400 nm is referred to as the NIR-II window) is highly transparent to biological soft tissues (700‒1400 nm)14 and has a large penetration depth; therefore, it provides deeper radiation penetration through tissue and blood. The photothermal agents play a crucial role in NIR laser-induced PTT, which requires the agents to possess the ability to harvest NIR light and transform the photon energy into heat energy. Photosensitive molecules (such as indocyanine green,15 malachite green,16 rhodamine 6G,16 etc.) are traditionally used as photothermal agents, but they are not ideal candidates because of their instability and lack of accumulation at tumor


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sites. To overcome these shortages, nanomaterials are currently being studied as substitutes for those photosensitive molecules because they have the distinct advantages of a higher stability against photobleaching and a better ability to accumulate at the tumor sites via passive targeting and/or active targeting by surface-assembled antibodies17, aptamers,10,11,18,19 and peptides.20 Typical examples of nanomaterials studied as PTT agents are noble metal nanoparticles,10,11,21-23 carbon materials,14,24 and metal-based sulfide nanoparticles.25,26 Au-based nanomaterials are, in particular, considered to be the best choice because of their tunable resonance characteristics, higher absorption ability in the NIR region, more efficient photothermal conversion, inert chemical activity in air, and more importantly, their excellent biocompatibility.8 However, for Au-based nanomaterials to be good PTT agents, their localized surface plasmon resonance (LSPR) absorption must be tuned to the NIR region. The LSPR absorption characteristics of Au nanomaterials are determined by several parameters including size, shape, structure, morphology, etc. Increasing the size of Au nanospheres (Au NSs) is the most facile approach to shift their LSPR absorption to a longer wavelength. However, this size-induced red-shift is very limited. For instance, the reported LSPR peaks of Au NSs with diameters of 5, 70, and 100 nm are located at approximately 520, 530, and 550 nm, respectively.

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Thus, several non-spherical Au

nanostructures such as Au nanorods,20,21,27 nanoshells,28,29 nanocages,30,31 nanoplates,32 and branched nanostructures33 have been synthesized and studied with the aim of shifting the LSPR peak positions of the Au-based nanomaterials. This is because altering the shape, morphology, and structure is a more effective means of shifting the LSRP bands than changing size. Although these non-spherical Au nanostructures can tune the LSPR absorption into the NIR region, their synthesis is not straightforward and usually needs tedious procedures. Moreover, they cannot be synthesized in scalable and reproducible quantities. One facile way to fully utilize the advantages of Au nanostructures and to tune their LSPR absorption to the NIR region is the elongation of the Au NSs along a single direction, which will give rise to electron oscillations parallel to the short and long axes and results in the transverse and longitudinal modes of the LSPR. For example, the LSPR absorption of the transverse mode of Au nanorods remains ACS Paragon Plus Environment

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at the same wavelength as that of the Au NSs, while its longitudinal mode has a significant red-shift into the NIR region. This work reports a facile way to synthesize tube-like Au nanostructures with a longitudinal LSPR mode. The synthesis of the tube-like Au nanostructures was achieved by assembly of Au NSs (~15 nm in diameter) through electrostatic interactions on the surface of attapulgite, which was used as a template. Attapulgite, a needle-shape nanometer-sized natural clay, is a hydrated magnesium silicate consisting of double chains of Si–O, which are further linked by a layer of octahedral magnesium atoms in 6-fold coordination, forming a network of strips that are joined together only along the edges.34-36 The natural clay has good biological compatibility and has been extensively used as a drug carrier, food additive, and enzyme catalyst support.34-36 The assembled Au NSs on the attapulgite surface link to each other and form a Au‒attapulgite nanocomposite with a unique structure, which behaves like an Au nanotube, showing both transverse and longitudinal modes of the LSPR. It should be emphasized that our approach to the synthesis of the tube-like Au‒attapulgite nanocomposite is very simple compared with those reported for Au nanorod preparations. For example, with the traditional template-directed synthesis of Au nanorods, where an anodic aluminum oxide membrane (AAO) is used as a template, the procedure involves coating a sacrificial metal (Ag or Cu) layer on one side of the AAO, electrodepositing Au into the pores of the AAO, dissolving the AAO, etc.37 Our tube-like Au‒ attapulgite nanocomposites could be readily obtained by simple electrostatic binding without the need for a sacrificial metal and without the removal of a template. The synthesized Au‒attapulgite nanocomposites exhibit high efficiency for the conversion of NIR light into heat energy and low cytotoxicity. A549 cells (human adenocarcinoma cell, NSCLC) were used to demonstrate the possibility of the nanocomposite as a PTT agent. The results showed that the Au‒attapulgite nanocomposite is efficient at killing the cancer cells and ablating tumors, which were constructed on the body of nude mice. Considering their facile synthesis, strong NIR longitudinal LSPR absorption, high photothermal conversion capabilities, and good biocompatibility, our synthesized nanocomposites have great potential to be used as a PTT agent in the clinic.


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RESULTS AND DISCUSSION Synthesis of the Au‒Attapulgite Nanocomposites. Attapulgite was used as the template for the synthesis of the Au–attapulgite nanocomposites. To allow for effective electrostatic binding of the negatively charged Au NSs on its surface, the surface charge of attapulgite was tuned to be positive by functionalizing it with differently charged polymers, which can tune the surface charge of attapulgite from approximately −28 mV to 45 mV (the procedures are detailed in the Experimental section). The polymer-coated attapulgite was then incubated with the negatively charged Au NSs, which have a size of ~15 nm in diameter (Figure S1) and a Zeta (ζ) potential of approximately –17 mV, to form the Au– attapulgite nanocomposites. The first indication of the formation of the Au–attapulgite nanocomposites was the alteration of the ζ value, which changed from ~45 mV (before incubation with Au NSs) to ~13 mV (after incubation with Au NSs). The formation of the Au–attapulgite nanocomposites can be further verified by transmission electron microscopic (TEM) images. The pure attapulgite (Figure 1A) has a needle-like morphology with an average size of 30 nm in width and 250‒500 nm in length. After assembly of Au NSs, there are a number of small Au NSs uniformly distributed on the attapulgite surface (Figure 1B), and these Au NSs link to each other along the attapulgite surface to form the tubelike Au–attapulgite nanocomposites (Figure 1C). The EDX spectrum depicted in Figure S2 shows the presence of Au, Si, O, and Mg elements. The lattice spacing of the assembled Au NSs is observed to be 0.236 nm from the high-resolution TEM (HRTEM) image (Figure 1D), which could be assigned to the (111) plane of Au. These results suggest the synthesis of the Au–attapulgite nanocomposites. X-ray diffraction (XRD) was used to investigate the structure of the synthesized Au–attapulgite nanocomposites (Figure 1E). The XRD pattern of attapulgite show peaks at 8.5, 13.9, 19.8, and 28.1º (curve a, Figure 1E), which are characteristic of pure attapulgite and correspond to the (110), (200), (040), and (400) planes, respectively. All of these diffraction peaks are the same as those of the reference pattern collected for attapulgite (JCPDS 29-0855). The XRD pattern of Au NSs (curve b, Figure 1E) shows the characteristic peaks of the (111), (200), (220), and (311) planes, indicating a face-


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centered cubic Au structure (JCPDS 4-0784). The XRD pattern of the Au–attapulgite nanocomposites reveals features of the attapulgite pattern and the Au NSs pattern (curve c, Figure 1E), further demonstrating the synthesis of the Au‒attapulgite nanocomposites by our simple method.

Figure 1. TEM images of pure attapulgite (A) and Au‒attapulgite nanocomposites (B, C). (D) HRTEM image of a Au NS assembled at the attapulgite surface. (E and F) XRD patterns (E) and UV–vis spectra (F) of pure attapulgite (a), Au NSs (b), and Au‒attapulgite nanocomposites (c). (G) The calculated results for the extinction (a), absorption (b), and scattering cross sections (c) of the synthesized Au– attapulgite nanocomposites with an inner diameter of 30 nm, outer diameter of 60 nm, and length of 430 nm. The inset shows the geometric parameters for the simulation.

After the synthesis of the Au‒attapulgite nanocomposites was confirmed, the UV–vis absorption characteristics of the nanocomposites were studied because the UV–vis absorption features are very important for a nanocomposite to be an effective PTT agent. The UV–vis spectrum of Au NSs shows a typical LSPR peak at 525 nm (curve b, Figure 1F), while that of pure attapulgite shows a featureless curve (curve a, Figure 1F). There are two absorption bands at ~535 and 670 nm appearing in the UV–vis spectrum of the Au‒attapulgite nanocomposites (curve c, Figure 1F). This feature is similar to that of Au nanorods,21,22 where the LSPR peak splits into two modes, e.g., the transverse and longitudinal modes, caused by the different orientations of the rod with respect to the electric field of the incident ACS Paragon Plus Environment

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light.38 This feature also suggests that the assembly of the Au NSs on the attapulgite surface is not simply aggregation to form large Au nanoparticles but is an assembly along the attapulgite surface to form a tube-like nanocomposite. The transverse mode of the Au‒attapulgite nanocomposites (centered at approximately 535 nm) is similar to the peak position observed for Au NSs (having a 10-nm red-shift that is due to a slight aggregation during Au NS assembly) because the transverse direction had a circular cross section profile similar to a sphere.8 The absorption band corresponding to the longitudinal mode, however, has shifted into the NIR region (centered at approximately 670 nm) with a large cross section. Therefore, we have successfully tuned the LSPR peak of Au NSs into the NIR region by simply assembling them onto the attapulgite surface and forming tube-like Au‒attapulgite nanocomposites. This is a simple approach to the synthesis of Au NS-based nanostructures with tailored LSPR in the NIR region. In addition to the LSPR peak position in the NIR, the high ratio of the absorption cross section (Cabs) relative to the scattering cross section (Csca) is also important to PTT agents. Therefore, the absorption and scattering spectra, together with extinction spectra of the tube-like Au‒attapulgite nanocomposite, were simulated by FDTD (Figure 1G), which is an explicit time marching algorithm used to solve Maxwell’s curl equations on a discrete spatial grid. In our calculations, we constructed a nanotubeshaped nanocomposite model composed of an inner 30-nm-diameter attapulgite coated with a 15-nmthick outer Au shell. The length of this tube-model is 430 nm, which refers to the mean length of the assynthesized nanocomposites. Of note, although the Au NSs link to each other on the attapulgite surface, they do not form a dense film, as was the case of the outer Au shell we constructed in the calculation mode. In the calculation mode, it is very difficult to construct a model in which the Au NSs on attapulgite are not continuous. For a simple and reasonable approximation, we use a nanotube-shaped nanocomposite model to simulate the absorption of the Au‒attapulgite nanocomposite. We considered the longitude mode, i.e., the incident beam is perpendicular to the long axis of the tube. It was observed that the extinction cross section consisted of absorption and scattering cross sections, and the absorption cross section was clearly larger than the scattering one. The calculated ratio of Cabs/Csca was 1.76, ACS Paragon Plus Environment

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indicating that a larger portion of the light absorption can be attributed to the extinction cross section of the synthesized Au‒attapulgite nanocomposites. As a result, the synthesized nanocomposites seem to be suitable for PTT applications as a photothermal conversion agent. Moreover, the amount of Au used is also significantly reduced compared with those reported for synthesizing pure Au nanostructures. The amount of Au in the Au–attapulgite nanocomposites is estimated to be ~13% of that used to synthesize pure Au nanotubes with a similar size. This is beneficial for practical clinical applications because Au is a noble metal with extremely low abundance in the earth’s crust (5 parts per billion).39 Thus, we expect the Au–attapulgite nanocomposites to be a good PTT agent for use in tumor therapy. Photothermal Performances of the Au–attapulgite Nanocomposites. Having synthesized and characterized the Au–attapulgite nanocomposites, their photothermal conversion capabilities were assessed. We irradiated the Au–attapulgite nanocomposites (100 µg mL‒1) using an 808-nm laser at a power density of 0.5 W cm‒2 and recorded the solution temperature as a function of exposure time. Au NS (480 µg mL‒1) and pure attapulgite suspensions (100 µg mL‒1), as well as PBS, were used as controls. A rapid temperature elevation in the case of the Au–attapulgite nanocomposites is observed, where the temperature increases from the ambient temperature to 53 ºC within 15 min (curve a, Figure 2A), whereas the Au NS and attapulgite suspensions are only heated to 35 and 31 ºC (curves b and c, Figure 2A), respectively, within the same irradiation time. For PBS, there is no obvious temperature rise after 15 min of irradiation (curve d, Figure 2A). Instead, the temperature is kept at 25‒28 ºC even with prolonged irradiation. These results suggest that the Au–attapulgite nanocomposites can rapidly absorb NIR light and efficiently convert light energy into thermal energy, implying that the synthesized tubelike Au–attapulgite nanocomposites have great potential as photothermal agents for use in PTT. The photothermal conversion characteristics of the Au–attapulgite nanocomposites were also verified by thermal images (Figure 2B). The temperature of the Au–attapulgite nanocomposite suspension rises rapidly and can reach more than 50 °C after it is irradiated with NIR light (image a, Figure 2B), while the temperature increase of the Au NSs, attapulgite, and PBS is negligible (images b, c, and d, Figure 2B), again demonstrating the high photothermal conversion ability of the synthesized nanocomposites. ACS Paragon Plus Environment

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Figure 2. (A) Temperature profiles as a function of irradiation time for Au–attapulgite nanocomposites (a, 100 µg mL‒1), Au NSs (b, 480 µg mL‒1), and attapulgite (c, 100 µg mL‒1), as well as PBS (d, 0.1 M). (B) Photothermal images of the suspensions of Au–attapulgite nanocomposites (a, 100 µg mL‒1), Au NSs (b, 480 µg mL‒1), and attapulgite (c, 100 µg mL‒1), as well as PBS (d, 0.1 M), after 15 min of irradiation. (C) Profiles of the temperature-irradiation time dependency for different concentrations of the Au–attapulgite nanocomposite suspension. The concentrations of the Au–attapulgite nanocomposite suspensions are 10 (a), 20 (b), 50 (c), 100 (d), and 200 µg mL‒1 (e). The results depicted in panels (A), (B), and (C) are obtained under irradiation at 808 nm with a power density of 0.5 W cm‒2. (D) Profiles of temperature-irradiation time dependency for different laser power densities. The concentration of the Au–attapulgite nanocomposite suspension is 100 µg mL‒1, and the power densities are 0.25 (a), 0.5 (b), 1.0 (c), and 1.5 W cm‒2 (d). (E) The course of the temperature of the Au–attapulgite nanocomposite suspension (100 mg mL‒1) under irradiation at 808 nm with a power density of 0.5 W cm‒2 for 15 min and then allowed to cool to room temperature after turning off the laser. (F) Plot of cooling time as a function of the negative natural logarithm of the temperature driving force obtained from a cooling stage, as shown in (E). The initial temperature is 25 °C.


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The photothermal performance of the Au–attapulgite nanocomposites can be affected by a variety of parameters, such as the concentration of nanocomposites, energy density of irradiation, irradiation time, etc., and this work examined these effects. When the irradiation power density is kept at 0.5 W cm–2, increasing the concentration of the Au–attapulgite nanocomposites in solution (0‒200 µg mL‒1) results in an enhancement of the temperature elevation rate (Figure 2C). The temperature can rise to ~53 ºC at an Au–attapulgite nanocomposite concentration of 100 µg mL‒1. The temperature can further rise to more than 70 ºC when the concentration of the nanocomposites is increased to 200 µg mL‒1. Because the cells will be destroyed if they are subjected to a temperature of 41‒47 ºC for tens of minutes, a Au– attapulgite nanocomposite concentration of 100 µg mL‒1 is used for further experiments. The irradiation power density also affects the heat performance, where a higher energy density induced a higher temperature elevation (Figure 2D). For example, there is a rapid rise in temperature to over 60 ºC when the light density is increased to over 1 W cm‒2. Considering the low skin tolerance threshold (~0.33 W cm‒2 at 808 nm40), an irradiation power density of 0.5 W cm‒2 is used when performing the photothermal experiments, which allows for enough hyperthermia (53 ºC) to kill cancer cells. To further demonstrate the photothermal conversion capabilities of the synthesized Au–attapulgite nanocomposites, the photothermal conversion efficiency (η), which is an important parameter for characterizing the PTT agents and plays a key role in the PTT, was estimated. A suspension of the Au– attapulgite nanocomposites (100 µg mL‒1) was irradiated with an 808-nm laser with a power density of 0.5 W cm‒2 until the suspension reached a steady-state temperature (within 15 min). Then, the laser was removed, and the suspension was allowed to naturally cool to the ambient temperature. A change in the course of the temperature plot with time was recorded and is depicted in Figure 2E, which shows that the temperature increases from 25.0 °C to a maximum temperature of 53 °C within 15 min of irradiation and then gradually decreases to ambient temperature after turning off the laser. According to the method proposed by Roper,41 the temperature data were fit to a linearized energy balance derived from a description of the microscale thermal dynamics in the suspension to determine the photothermal conversion efficiency (η), which was calculated with the following equation (1): ACS Paragon Plus Environment

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=

∙ ∙

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(1)

where h is the heat transfer coefficient of the nanocomposites, A is the area cross section of irradiation, Q0 is the heat dissipated from light absorbed by the quartz sample cell and water (this was measured independently as 25.7 mW using borosilicate glass cells containing water without the addition of the Au–attapulgite nanocomposites), I is the incident laser power, A808 is the absorbance of the Au‒ attapulgite nanocomposites at 808 nm, Tmax is the highest temperature that can be reached under irradiation, and Tsurr is the surrounding temperature. The value of h·A can be derived from the following equation (2): =

∑ , ∙

(2)

where mi and Cp,i are the mass of and heat capacity of the irradiated system, respectively, including water, the quartz cell, and the Au–attapulgite nanocomposites. The values for mi are 0.5 and 5.7 g, while the values for Cp,i are 4.2 J g–1 K–1 and 0.89 J g–1 K–1 for water and the quartz cell,42,43 respectively. The time constant, τs, is defined as the slope of cooling time against –Ln(θ) (Figure 2F), where θ is the temperature driving force, which is defined by equation (3): !=

"#$

(3)

where T is the temperature of the system. The value of η for the Au–attapulgite nanocomposites was calculated as 25.6%, which is much higher than that of Au NSs with a diameter of ~15 nm (6.5%) (Figure S3) and is slightly higher than that of commercial Au nanorods (21%, 23 × 7 nm with an aspect ratio of 3.3).44 The high value of η for the Au–attapulgite nanocomposites is probably due to their LSPR absorption band (longitude mode) in the NIR region and the larger extinction cross section, together with the large ratio of Cads/Csca (Figures 1F and G). Although the η value of the Au–attapulgite nanocomposites is not very high—it is even lower than some polymer or polymer-coated oxide PTT agents, such as dopamine-melanin colloidal nanospheres (~40%)45 and polypyrrole (PPy)-coated Fe3O4 nanoparticles (~49.0%)46 —it is sufficient to generate enough hyperthermia to kill cancer cells (see the results depicted below). ACS Paragon Plus Environment

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In Vitro and in Vivo Photothermal Studies. To further demonstrate the possibility of the assynthesized Au–attapulgite nanocomposites to be used as a new PTT agent, we evaluated the PTT effects of the nanocomposites on A549 cells and tumor-bearing nude mice. The cytotoxicity of the Au– attapulgite nanocomposites was firstly estimated by an MTT assay. The results indicated that the cellular viability of the A549 cells remained at more than 92%, even when the cells were incubated with Au–attapulgite nanocomposites (the concentrations of the nanocomposites range from 0 to 200 µg mL‒1) for 12 h (Figure S4), indicating the low cytotoxicity of the nanocomposites within this concentration range. Then, the cellular uptake of the Au–attapulgite nanocomposites by A549 cells was estimated because the cellular uptake of a photothermal agent is an important parameter to evaluate its therapeutic effect. For this estimation, ~104 A549 cells were incubated with 100 µg mL‒1 Au–attapulgite nanocomposites for 12 h at 37 ºC, followed by PBS washing to remove unbound nanocomposites. Then, the cellular uptake of the Au–attapulgite nanocomposites was evidenced by changes in the features of the UV–vis spectrum. A featureless curve was observed for the unadulterated A549 cells in the NIR region, while an absorption peak centered at ~810 nm was observed after the cells were incubated with the Au–attapulgite nanocomposites (Figure S5), indicative of the cellular uptake of the nanocomposites. The amount of cellular uptake of the nanocomposites was quantified by ICP measurement, which showed that there are approximately 8.2 fg Au per cell after a 12-h incubation, indicating the efficient uptake of the Au–attapulgite nanocomposites by A549 cells. The PTT effects of the Au–attapulgite nanocomposites on A549 cells were studied by first incubating the cells with the nanocomposites (100 µg mL‒1) for 12 h and then irradiating the cells with an 808-nm laser at 0.5 W cm‒2 for 0‒15 min. The numbers of the live and dead cells were directly observed under a fluorescence microscope after the cells were treated by live-dead staining. As shown in Figure 3A, the cells treated with the nanocomposites show significant death (the live cells emitted the green emission, and the dead ones emitted the red emission) upon irradiation, and the number of dead cells increases with prolonging irradiation time (images a–e). Approximately all of the cells die after a 15-min irradiation (image e). However, a few cells did die in the control group (the cells were not incubated ACS Paragon Plus Environment

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with the Au–attapulgite nanocomposites) after being irradiated by a laser for 15 min; moreover, the number of dead cells is almost invariant with irradiation time (images a'–e'). These results indicate that the synthesized Au–attapulgite nanocomposites have a good PTT effect on the A549 cells.

Figure 3. (A) Fluorescence images of live-dead stained A549 cells incubated with (a‒e) and without (a'‒e') the Au–attapulgite nanocomposite suspension (100 µg mL‒1) under laser irradiation at 808 nm with a power of 0.5 W cm‒2 for 0‒15 min. The laser irradiation time is 0 (a, a'), 2 (b, b'), 5(c, c'), 10 (d, ACS Paragon Plus Environment

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d'), and 15 min (e, e'). (B) The cell viability of A549 cells incubated with (a) and without (b) the Au– attapulgite nanocomposite suspension (100 µg mL‒1) under laser irradiation at 808 nm with a power density of 0.5 W cm‒2 for various lengths of time.

We also performed MTT measurements to confirm the PTT effects of the Au–attapulgite nanocomposites on the A549 cells. The results depicted in Figure 3B show that the viability of cells incubated with the nanocomposites decreases rapidly with irradiation time, and the viability is almost zero after 15 min of irradiation (curve a in Figure 3B). Yet, the viability decreases very slowly with irradiation time for those cells not incubated with the nanocomposites; the viability is still more than 90% even after irradiation for 15 min (curve b in Figure 3B). These results are in good agreement with those observed from fluorescence microscopy (Figure 3A), further indicating the promising PTT effects of the synthesized nanocomposites on the cancer cells. We further performed photothermal ablation of tumors borne on nude mice to evaluate the in vivo PTT efficiency of the Au–attapulgite nanocomposites. A549 cell-bearing BALB/c nude mice were divided into four groups (groups I–IV), and each group contained three mice. Group IV is the control group, and the mice in this group were allowed to grow naturally and without any treatment. Groups I, II, and III are treatment groups. The mice in groups I and II were first intratumorally injected with 100 µL of the Au–attapulgite nanocomposite suspension (1 mg mL‒1) and PBS, respectively, and then irradiated at the tumor site using an 808-nm laser with a power of 0.5 W cm‒2 for 15 min. The mice in group III were also injected with 100 µL of the Au–attapulgite nanocomposite suspension (1 mg mL‒1), but they were not treated by laser irradiation. The temperature at the tumor site was monitored with an MG33 photothermal therapy monitoring system by recording the infrared thermal images (Figure 4A) and plotting them against the irradiation time (Figure 4B). The temperature increases rapidly after irradiation of the tumor site that had been injected with the Au–attapulgite nanocomposite suspension (images a–e in Figure 4A) and can reach approximately 50 ºC (curve a in Figure 4B). However, the temperature at the tumor site that had been injected with PBS increases slowly (images a'–e' in Figure 4A), and the extent of the temperature increase is low. The temperature can only be increased to 34 ºC, ACS Paragon Plus Environment

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as shown by curve b in Figure 4B. These results suggest the great ability of the nanocomposites to convert light energy into heat energy and their potential usefulness as an in vivo PTT agent.

Figure 4. (A) Photothermal images of the mice in group I (injection of Au–attapulgite nanocomposites) (a–e) and group II (injection of PBS) (a'–e') with an irradiation time of 15 min. (B) Temperature profiles as a function of irradiation time for the tumor site of mice in group I (a) and group II (b). (C–E) Typical images of the tumor sizes (C), tumor volumes (D), and pathological slices of the tumor tissue (E) of the ACS Paragon Plus Environment

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mice in groups I (a), II (b), III (c), and IV (d). V (in mm3) is the tumor volume after the treatment, and V0 is the original tumor volume before the treatment. The data shown here for groups I and II were obtained after an 808-nm laser irradiation with a power density of 0.5 W cm‒2 for 15 min. The scale bar in slices a–d is 20 µm.

After laser irradiation, the tumors of mice in group I were found to be effectively ablated, leaving black scars at the tumor sites (image a, Figure 4C). In contrast, the tumor sizes of the mice in groups II (image b, Figure 4C) and III (image c, Figure 4C) were almost the same as those in the control group (image d, Figure 4C). The average tumor volume was also continuously monitored for the next 10 days, starting from the irradiation (Figure 4D). It is found that tumor volumes for the mice in group I (curve a, Figure 4D) are only 10% of those in group IV (curve d, Figure 4D). However, the tumor volumes of the mice in groups II (curve b, Figure 4D) and III (curve c, Figure 4D) were found to be almost the same as those in group IV. The tumor volumes did not regrow for the mice in group I in the following days (curve a, Figure 4D). Moreover, no significant body weight drop was noticed during this period of observation (Figure S6), suggesting that it had been effectively inhibited after irradiation. However, the tumor volumes in groups II and III grew continuously and were the same as those for the mice in group IV. To test whether the PTT ablation of the tumors contributed to the inhibition of cancer cell metastasis, a pathological examination by hematoxylin and eosin (HE) staining was performed, and the results are depicted in Figure 4E. The tumor tissue sections of the mice in group I show a significant decrease in pathological mitosis and mucous secretion of cancer cells (slice a, Figure 4E) because we cannot see any obvious cancer cell divisions. Moreover, the cell sizes and morphologies are approximately uniform (marked with the green circle in slice a), and the organized tissue structures are also clearly observed, suggesting that cancer cell metastasis has been effectively inhibited. However, the slices from groups II (slice b, Figure 4E) and III (slice c, Figure 4E) do not exhibit any tumor necrosis, which is the same as the results from group IV (slice d, Figure 4E). The pathological mitosis (or cell division) of the cancer cells is clearly observed (marked with red circles in slices b–d), suggesting that the cancer cells grow abnormally. The cell heterogeneities are clear (the cell sizes and morphologies are not uniform, as ACS Paragon Plus Environment

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highlighted by those cells in the green circles in slices b–d), and the tissue structure is in disorder, implying that the cancer cells are growing rapidly. These results indicate the high efficiency of the assynthesized nanocomposites as a PTT agent for tumor therapy. To further evaluate the biosafety of the Au–attapulgite nanocomposites, their concentration at the tumor site was monitored. The tumors were collected at different times (1, 6, 12, 24, and 48 h) after injection of Au–attapulgite nanocomposites, were dissolved with 5 mL of aqua regia, and were heated at 60 °C for 12 h to remove the aqua regia. Caution: aqua regia is extremely dangerous and should be handled with extreme caution. Gloves and eye protection are required for handling. After that, the solutions were centrifuged and the supernatants were kept for further ICP analysis. As shown in Figure S7, the concentration of Au–attapulgite nanocomposites at the tumor site gradually decreases with time and reaches a negligible level after 48 h, suggesting that the nanocomposites can be excreted from the mice. The reason for the rapid metabolism of the nanocomposites may be ascribed to their efficient dissociation into individual small Au NSs after irradiation (Figure S8), which will facilitate the clearance process in the body.47 All of these fascinating properties possessed by the synthesized Au– attapulgite nanocomposites indicate their need to be further studied with other different tumor models to find possible clinical applications. EXPERIMENTAL SECTION Synthesis of Au‒attapulgite Nanocomposites. To prepare the Au‒attapulgite nanocomposites, we synthesized Au NSs based on published procedures48 with a minor modification. Typically, in a 1-L condenser-equipped round-bottom flask, 100 mL of 1 mM HAuCl4 was first brought to a rolling boil with vigorous stirring. Then, 10 mL of 38.8 mM sodium citrate was rapidly added to this solution, resulting in a color change from pale yellow to burgundy. Boiling was continued for 10 min, the heating mantle was removed, and stirring was continued for an additional 15 min. After the solution was cooled to room temperature, it was filtered through a 0.8-µm nylon membrane filter. Transmission electron microscopic (TEM) images and dynamic light scattering (DLS) analysis indicated that the synthesized ACS Paragon Plus Environment

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Au NSs had a size of ~15 nm in diameter (Figure S1). The Zeta (ζ) potential of the prepared Au NSs was measured to be approximately –17 mV. Prior to assembly of the synthesized Au NSs on the surface of attapulgite, attapulgite (Jiuchuan Clay Company, Xuyi, China) was treated according to previously reported procedures.36 In brief, it was ground in a KM-10 planetary mill (Nanjing University Instrument Plant, China) at 800 rpm for 2 h. The ground sample (1 g) was then treated with 100 mL of HCl (8 M) at 80 °C for 2 h and cooled to ambient temperature. The suspension was filtered through a 0.65-µm microporous nylon membrane and thoroughly washed with double distilled water. The product was dried at 80 °C for 12 h in air to obtain the purified sample (white powder). The assembly of Au NSs on the attapulgite surface was based on electrostatic interactions. The isoelectric point of attapulgite is 4–4.5,34 implying that it has an overall negative charge surface in pH 7.4 PBS. This is verified by its ζ value (measured to be approx. –28 ± 2 mV). To favor the assembly of the negatively charged Au NSs on the attapulgite surface, we tuned the attapulgite surface from a negative charge to a positive charge by the deposition of positively charged poly(dimethyl diallyl ammonium chloride) (PDDA, Mw = 100,000‒200,000, Sigma) and negatively charged poly(methacrylic acid) (PMAA, Mw = 9,500, Sigma) in turns using the layer-by-layer (LBL) assembly technique. Firstly, 10 mL of a PDDA aqueous solution (1%) was added to 1 mL of the attapulgite suspension (1 mg mL–1) and stirred for 30 min. The PDDA-coated attapulgite particles were collected by centrifuging, which was followed by washing three times with water. Then, they were re-dispersed in 1 mL of water and ultrasonicated for 5 min to prevent aggregation. Negatively charged PMAA was then deposited onto the surface of the above PDDA-coated attapulgite by a similar procedure. We added 10 mL of a PMAA aqueous solution (1%) to 1 mL of the PDDA-coated attapulgite suspension (1 mg mL–1) and stirred for 30 min to form a negatively charged outermost sphere. Then, the suspension was treated by centrifuging, washing and ultrasonicating. Finally, another 10 mL of the PDDA aqueous solution (1%) was added to 1 mL of the above obtained polymer-coated attapulgite suspension (1 mg mL–1). After being treated by the same procedure, the positively charged attapulgite particles were obtained. ACS Paragon Plus Environment

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The positively charged attapulgite particles were mixed with Au NSs to form Au‒attapulgite nanocomposites. Then, 1 mL of the polymer-coated attapulgite particles (1 mg mL–1) was added to 30 mL of the negatively charged Au NSs and stirred overnight to allow for the adequate assembly of Au NSs on the attapulgite surface. The obtained Au‒attapulgite nanocomposites were centrifuged, washed three times with water to remove the excess Au NSs, and subsequently dispersed into 1 mL PBS by ultrasonication for further use. Ultrasonication at power lower than 50 W (28 kHz) does not cause the loss of the Au NSs from the surface of attapulgite). The suspension of the Au‒attapulgite nanocomposites (1 mg mL–1) remains homogeneous with a clear pale wine red color for several days (at least one week) at ambient temperature without any perceptible aggregation or color change. The polydispersity index (PDI) was estimated to be ~0.43 based on dynamic light scattering (DLS) measurements, which were performed on a Zeta potential analyzer (Nano Z, Malvern). Of note, the PDI of the attapulgite is ~0.35. Instruments. The transmission electron microscopic (TEM) and high-resolution TEM (HRTEM) images were acquired on a JEM–2100F transmission electron microscope. X–ray diffraction (XRD) patterns were recorded using a Rigaku/Max–3A X–ray diffractometer with Cu Kα radiation (λ = 0.15418 nm). UV‒Vis spectroscopy measurements were performed on a Cary 5000 UV‒Vis‒NIR spectrometer (Varian). ICP measurements were carried out on an Optima 7300 DV (Perkin Elmer). The ζ potential measurement and dynamic light scattering (DLS) analysis were carried out on a Zetasizer Nano ZS90 Analyzer (Malvern). Simulation Details. Three-dimensional finite-difference time-domain (FDTD) simulations from Lumerical Solutions, Inc. (Vancouver, Canada) are performed to calculate the scattering and absorption cross sections. The boundary conditions of the simulation domain are perfectly matched with the layer absorbing boundaries. We considered the longitude mode, i.e., the incident beam is perpendicular to the long axis of the tube. The calculation region is 0.5 × 0.5 × 0.5 µm3, where the grid resolution was set to be 2 nm. A cylindrically symmetrical Au nanotube with an inner diameter of 30 nm, an outer diameter of 60 nm, and a length of 430 nm was implemented in the cell. The optical constant of the dielectric ACS Paragon Plus Environment

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permittivity with wavelength was adapted from the Johnson and Christy database attached with the FDTD.49 The refractive index of the surrounding medium was set to be 1.33 for water, and the refractive constant of attapulgite was set as 1.50. In Vitro Cell Uptake Study. MTT assays were performed to evaluate the cytotoxicity of the Au‒ attapulgite nanocomposites using A549 cells (detailed procedures on cell culture and the MTT assay are presented in the SI). To quantify the cellular uptake of Au‒attapulgite nanocomposites, ICP (optical emission spectroscopy using an inductively coupled plasma as the ionization source) measurements were carried out. A549 cells (~1 × 104) were incubated with 100 µg mL‒1 Au‒attapulgite nanocomposites for 12 h, digested by 0.25% trypsin containing 0.02% EDTA, centrifuged for 8 min at 1500 rpm, and washed three times with water. Subsequently, samples were treated with aqua regia to dissolve Au. The resulting solutions were left standing at room temperature for 6 h and then heated at a temperature of 60 ºC for 12 h to remove the aqua regia. The residuals were dissolved in deionized water and determined using ICP. In Vitro NIR Photothermal Therapy of A549 cells. After incubation with Au‒attapulgite nanocomposites (100 µg mL‒1) for 12 h and rinsing with PBS, the A549 cells were exposed to irradiation with a wavelength of 808 nm at different power densities (0.25‒1.5 W cm‒2) for various times (0‒15 min). Then, the cells were stained by live‒dead kits (Nanjing KeyGen Biotech. Ltd.) to evaluate their viability. The fluorescence images were captured with an AXIO microscope (AxioObserver A1, Carl Zeiss) equipped with Epi–fluorescence and an AxioCam MRc imaging system using fluorescence mode. Living cells showed a green color, and dead ones exhibited a red color. Therefore, the cell viability can be qualitatively observed from the color of the cells. In Vivo Photothermal Therapy of Tumor-Bearing Mice. BALB/c nude mice (~20 g body weight) bearing A549 cells (6 weeks) were obtained from Nanjing KeyGen Biotech. Ltd. Twelve mice with a tumor size of 80 mm3 were randomly divided into four groups: groups I–IV. Each group contained three mice. Groups I, II, and III are treatment groups. Group IV is a control group, and the mice in this group were allowed to grow naturally without any treatment. The mice in groups I–III were first anesthetized ACS Paragon Plus Environment

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using trichloroacetaldehyde hydrate (10%) at a dose of 40 mg kg–1 body weight while being maintained at normal body temperature. The mice in groups I and II were first injected with the Au‒attapulgite nanocomposites (100 µL, 1 mg mL‒1) and PBS, respectively, at the tumor site located at a depth of ~4 mm (the dose is 5 mg kg‒1 body weight). Then, the tumor site was irradiated using an 808-nm laser with a power of 0.5 W cm‒2 for 15 min. The mice in group III were also injected with 100 µL of the Au– attapulgite nanocomposite suspension (1 mg mL‒1), but they were not treated by laser irradiation. Prior to the injection, the Au‒attapulgite nanocomposites were disinfected. To monitor the temperature changes at the tumor site during irradiation, infrared thermal images were recorded with a photothermal therapy monitoring system MG33 (Shanghai Magnity Electronics Co. Ltd.). Tumor growth and mouse weight were measured over the following 10 days. When the experiments were finished, the tumors were removed and their volume was estimated (V, mm3) using the following formula: V = a × b2/2, where a is the length and b is the width in millimeters. Relative tumor volumes were calculated as V/V0, where V0 is the original tumor volume before the treatment. Then, the tumors were embedded in paraffin and sectioned into 4-µm slices. The slides were stained by hematoxylin/eosin and observed under an AXIO microscope to evaluate the PTT effects on the tumors. CONCLUSIONS We have reported a facile method to synthesize the tube-like Au‒attapulgite nanocomposites by electrostatic binding of Au NSs to the surface of a nanosized mineral, attapulgite. This tube-like Au‒ attapulgite nanocomposite, consisting of numerous Au NSs linked to each other along the attapulgite surface, exhibits a longitudinal absorption mode shifted to the NIR region (approximately 670 nm). They have a high ability to convert photon energy into heat energy and possess a great Cabs/Csca ratio with a photothermal conversion efficiency of 25.6%. This nanocomposite can be effectively taken up by cells and possesses good biocompatibility, which is evidenced by the cell viability remaining at more than 92% even when the cells were incubated with nanocomposite concentrations as high as 200 µg mL‒ 1

. The in vitro and in vivo photothermal therapy results indicate that the Au‒attapulgite nanocomposites ACS Paragon Plus Environment

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can be used as effective PTT agents because the model cancer cells (A549 cells) almost died after they were incubated with the nanocomposites (at 100 µg mL–1) for 12 h and irradiated by an 808-nm laser with a power density of 0.5 W cm–2 for 15 min. Moreover, the tumors on nude mice can also be effectively ablated without regrowth of the tumors during the following period of observation (at least 10 days). Considering their ease of synthesis, the strong NIR longitudinal LSPR absorption, high photothermal conversion capabilities, good biocompatibility, and effective PTT results, the tube-like Au‒attapulgite nanocomposites hold great potential to be a new PTT agent for use in the clinic. ASSOCIATED CONTENT Supporting Information The detailed procedures of the cell culture and biocompatibility assay; TEM images and DLS analysis of Au NSs; EDX spectrum of the synthesized Au‒attapulgite nanocomposites; photothermal conversion behavior of Au NSs; cytotoxicity evaluation of the as-synthesized nanocomposites by the MTT assay; UV–vis spectrum of the A549 cells before and after incubation with the nanocomposites; the body weight of mice after PTT; and a TEM image of the synthesized nanocomposites after irradiation. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGEMNETS This work is supported by the NSFC (21175067, 21273117, 21375063, 21335004, and 21405083), NSF of the Jiangsu Higher Education Institutions (14KJB150012), and Priority Academic Program Development of Jiangsu Higher Education Institutions.


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